OPTIMIZED Fc VARIANTS

ABSTRACT

The present invention relates to Fc variants having decreased affinity for FcγRIIb, methods for their generation, Fc polypeptides comprising optimized Fc variants, and methods for using optimized Fc variants.

This application is a continuation of Ser. No. 12/896,610, filed Oct. 1,2010, which is a continuation of U.S. application Ser. No. 11/24,620,filed May 5, 2005, which claims benefit under 35 U.S.C. §119(e) to U.S.Ser. No. 60/568,440, filed May 5, 2004; 60/589,906 filed Jul. 20, 2004;60/627,026 filed Nov. 9, 2004; 60/626,991 filed Nov. 10, 2004;60/627,774 filed Nov. 12, 2004.

FIELD OF THE INVENTION

The present invention relates to novel optimized Fc variants,engineering methods for their generation, and their application,particularly for therapeutic purposes.

BACKGROUND OF THE INVENTION

Antibodies are immunological proteins that bind a specific antigen. Inmost mammals, including humans and mice, antibodies are constructed frompaired heavy and light polypeptide chains. Each chain is made up ofindividual immunoglobulin (Ig) domains, and thus the generic termimmunoglobulin is used for such proteins. Each chain is made up of twodistinct regions, referred to as the variable and constant regions. Thelight and heavy chain variable regions show significant sequencediversity between antibodies, and are responsible for binding the targetantigen. The constant regions show less sequence diversity, and areresponsible for binding a number of natural proteins to elicit importantbiochemical events. In humans there are five different classes ofantibodies including IgA (which includes subclasses IgA1 and IgA2), IgD,IgE, IgG (which includes subclasses IgG1, IgG2, IgG3, and IgG4), andIgM. The distinguishing features between these antibody classes aretheir constant regions, although subtler differences may exist in thevariable region. FIG. 1 shows an IgG1 antibody, used here as an exampleto describe the general structural features of immunoglobulins. IgGantibodies are tetrameric proteins composed of two heavy chains and twolight chains. The IgG heavy chain is composed of four immunoglobulindomains linked from N- to C-terminus in the orderV_(H)—C_(H)1-C_(H)2-C_(H)3, referring to the variable heavy domain,constant heavy domain 1, constant heavy domain 2, and constant heavydomain 3. The IgG C_(H)1, C_(H)2, and C_(H)3 domains are also referredto as constant gamma 1 domain (Cγ1), constant gamma 2 domain (Cγ2), andconstant gamma 3 domain (Cγ3) respectively. The IgG light chain iscomposed of two immunoglobulin domains linked from N- to C-terminus inthe order V_(L)-C_(L), referring to the light chain variable domain andthe light chain constant domain respectively.

The variable region of an antibody contains the antigen bindingdeterminants of the molecule, and thus determines the specificity of anantibody for its target antigen. The variable region is so named becauseit is the most distinct in sequence from other antibodies within thesame class. The majority of sequence variability occurs in thecomplementarity determining regions (CDRs). There are 6 CDRs total,three each per heavy and light chain, designated V_(H) CDR1, V_(H) CDR2,V_(H) CDR3, V_(L) CDR1, V_(L) CDR2, and V_(L) CDR3. The variable regionoutside of the CDRs is referred to as the framework (FR) region.Although not as diverse as the CDRs, sequence variability does occur inthe FR region between different antibodies. Overall, this characteristicarchitecture of antibodies provides a stable scaffold (the FR region)upon which substantial antigen binding diversity (the CDRs) can beexplored by the immune system to obtain specificity for a broad array ofantigens. A number of high-resolution structures are available for avariety of variable region fragments from different organisms, someunbound and some in complex with antigen. The sequence and structuralfeatures of antibody variable regions are well characterized (Morea etal., 1997, Biophys Chem 68:9-16; Morea et al., 2000, Methods 20:267-279,incorporated by reference), and the conserved features of antibodieshave enabled the development of a wealth of antibody engineeringtechniques (Maynard et al., 2000, Annu Rev Biomed Eng 2:339-376,incorporated by reference). For example, it is possible to graft theCDRs from one antibody, for example a murine antibody, onto theframework region of another antibody, for example a human antibody. Thisprocess, referred to in the art as “humanization”, enables generation ofless immunogenic antibody therapeutics from nonhuman antibodies.Fragments comprising the variable region can exist in the absence ofother regions of the antibody, including for example the antigen bindingfragment (Fab) comprising V_(H)—Cγ1 and V_(H)-C_(L), the variablefragment (Fv) comprising V_(H) and V_(L), the single chain variablefragment (scFv) comprising V_(H) and V_(L) linked together in the samechain, as well as a variety of other variable region fragments (Littleet al., 2000, Immunol Today 21:364-370, incorporated by reference).

The Fc region of an antibody interacts with a number of Fc receptors andligands, imparting an array of important functional capabilitiesreferred to as effector functions. For IgG the Fc region, as shown inFIG. 1, comprises Ig domains Cγ2 and Cγ3 and the N-terminal hingeleading into Cγ2. An important family of Fc receptors for the IgG classare the Fc gamma receptors (FcγRs). These receptors mediatecommunication between antibodies and the cellular arm of the immunesystem (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220;Ravetch et al., 2001, Annu Rev Immunol 19:275-290). In humans thisprotein family includes FcγRI (CD64), including isoforms FcγRIa, FcγRIb,and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (includingallotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2),and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65,incorporated by reference). These receptors typically have anextracellular domain that mediates binding to Fc, a membrane spanningregion, and an intracellular domain that may mediate some signalingevent within the cell. These receptors are expressed in a variety ofimmune cells including monocytes, macrophages, neutrophils, dendriticcells, eosinophils, mast cells, platelets, B cells, large granularlymphocytes, Langerhans' cells, natural killer (NK) cells, and γδ Tcells. Formation of the Fc/FcγR complex recruits these effector cells tosites of bound antigen, typically resulting in signaling events withinthe cells and important subsequent immune responses such as release ofinflammation mediators, B cell activation, endocytosis, phagocytosis,and cytotoxic attack. The ability to mediate cytotoxic and phagocyticeffector functions is a potential mechanism by which antibodies destroytargeted cells. The cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell is referred to as antibodydependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, AnnuRev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275-290,incorporated by reference). The cell-mediated reaction whereinnonspecific cytotoxic cells that express FcγRs recognize bound antibodyon a target cell and subsequently cause phagocytosis of the target cellis referred to as antibody dependent cell-mediated phagocytosis (ADCP).A number of structures have been solved of the extracellular domains ofhuman FcγRs, including FcγRIIa (pdb accession code 1H9V) (Sondermann etal., 2001, J Mol Biol 309:737-749) (pdb accession code 1 FCG) (Maxwellet al., 1999, Nat Struct Biol 6:437-442), FcγRIIb (pdb accession code2FCB) (Sondermann et al., 1999, Embo J 18:1095-1103); and FcγRIIIb (pdbaccession code 1E4J) (Sondermann et al., 2000, Nature 406:267-273,incorporated by reference). All FcγRs bind the same region on Fc, at theN-terminal end of the Cγ2 domain and the preceding hinge, shown in FIG.2. This interaction is well characterized structurally (Sondermann etal., 2001, J Mol Biol 309:737-749 incorporated by reference), andseveral structures of the human Fc bound to the extracellular domain ofhuman FcγRIIIb have been solved (pdb accession code 1E4K) (Sondermann etal., 2000, Nature 406:267-273) (pdb accession codes 1IIS and 1IIX)(Radaev et al., 2001, J Biol Chem 276:16469-16477, incorporated byreference), as well as has the structure of the human IgE Fc/FcεRIαcomplex (pdb accession code 1F6A) (Garman et al., 2000, Nature406:259-266, incorporated by reference).

The different IgG subclasses have different affinities for the FcγRs,with IgG1 and IgG3 typically binding substantially better to thereceptors than IgG2 and IgG4. All FcγRs bind the same region on IgG Fc,yet with different affinities: the high affinity binder FcγRI has a Kdfor IgG1 of 10⁻⁸ M⁻¹, whereas the low affinity receptors FcγRII andFcγRIII generally bind at 10⁻⁶ and 10⁻⁵ respectively. The extracellulardomains of FcγRIIIa and FcγRIIIb are 96% identical, however FcγRIIIbdoes not have a intracellular signaling domain. Furthermore, whereasFcγRI, FcγRIIa/c, and FcγRIIIa are positive regulators of immunecomplex-triggered activation, characterized by having an intracellulardomain that has an immunoreceptor tyrosine-based activation motif(ITAM), FcγRIIb has an immunoreceptor tyrosine-based inhibition motif(ITIM) and is therefore inhibitory. Thus the former are referred to asactivation receptors, and FcγRIIb is referred to as an inhibitoryreceptor. The receptors also differ in expression pattern and levels ondifferent immune cells. Yet another level of complexity is the existenceof a number of FcγR polymorphisms in the human proteome. A particularlyrelevant polymorphism with clinical significance is V158/F158 FcγRIIIa.Human IgG1 binds with greater affinity to the V158 allotype than to theF158 allotype. This difference in affinity, and presumably its effect onADCC and/or ADCP, has been shown to be a significant determinant of theefficacy of the anti-CD20 antibody rituximab (Rituxan®, a registeredtrademark of IDEC Pharmaceuticals Corporation). Patients with the V158allotype respond favorably to rituximab treatment; however, patientswith the lower affinity F158 allotype respond poorly (Cartron et al.,2002, Blood 99:754-758, incorporated by reference). Approximately 10-20%of humans are V158N158 homozygous, 45% are V158/F158 heterozygous, and35-45% of humans are F158/F158 homozygous (Lehrnbecher et al., 1999,Blood 94:4220-4232; Cartron et al., 2002, Blood 99:754-758, incorporatedby reference). Thus 80-90% of humans are poor responders, that is theyhave at least one allele of the F158 FcγRIIIa.

An overlapping but separate site on Fc, shown in FIG. 1, serves as theinterface for the complement protein C1q. In the same way that Fc/FcγRbinding mediates ADCC, Fc/C1q binding mediates complement dependentcytotoxicity (CDC). C1q forms a complex with the serine proteases C1rand C1s to form the C1 complex. C1q is capable of binding sixantibodies, although binding to two IgGs is sufficient to activate thecomplement cascade. Similar to Fc interaction with FcγRs, different IgGsubclasses have different affinity for C1q, with IgG1 and IgG3 typicallybinding substantially better to the FcγRs than IgG2 and IgG4. There iscurrently no structure available for the Fc/C1q complex; however,mutagenesis studies have mapped the binding site on human IgG for C1q toa region involving residues D270, K322, K326, P329, and P331, and E333(Idusogie et al., 2000, J Immuno/164:4178-4184; Idusogie et al., 2001, JImmuno/166:2571-2575, incorporated by reference).

A site on Fc between the Cγ2 and Cγ3 domains, shown in FIG. 1, mediatesinteraction with the neonatal receptor FcRn, the binding of whichrecycles endocytosed antibody from the endosome back to the bloodstream(Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie etal., 2000, Annu Rev Immunol 18:739-766, incorporated by reference). Thisprocess, coupled with preclusion of kidney filtration due to the largesize of the full length molecule, results in favorable antibody serumhalf-lives ranging from one to three weeks. Binding of Fc to FcRn alsoplays a key role in antibody transport. The binding site for FcRn on Fcis also the site at which the bacterial proteins A and G bind. The tightbinding by these proteins is typically exploited as a means to purifyantibodies by employing protein A or protein G affinity chromatographyduring protein purification. Thus the fidelity of this region on Fc isimportant for both the clinical properties of antibodies and theirpurification. Available structures of the rat Fc/FcRn complex (Martin etal., 2001, Mol Cell 7:867-877, incorporated by reference), and of thecomplexes of Fc with proteins A and G (Deisenhofer, 1981, Biochemistry20:2361-2370; Sauer-Eriksson et al., 1995, Structure 3:265-278; Tashiroet al., 1995, Curr Opin Struct Biol 5:471-481, incorporated byreference) provide insight into the interaction of Fc with theseproteins.

A key feature of the Fc region is the conserved N-linked glycosylationthat occurs at N297, shown in FIG. 1. This carbohydrate, oroligosaccharide as it is sometimes referred, plays a critical structuraland functional role for the antibody, and is one of the principlereasons that antibodies must be produced using mammalian expressionsystems. While not wanting to be limited to one theory, it is believedthat the structural purpose of this carbohydrate may be to stabilize orsolubilize Fc, determine a specific angle or level of flexibilitybetween the Cγ3 and Cγ2 domains, keep the two Cγ2 domains fromaggregating with one another across the central axis, or a combinationof these. Efficient Fc binding to FcγR and C1q requires thismodification, and alterations in the composition of the N297carbohydrate or its elimination affect binding to these proteins (Umañ aet al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, BiotechnolBioeng 74:288-294; Mimura et al., 2001, J Biol Chem 276:45539-45547;Radaev et al., 2001, J Biol Chem 276:16478-16483; Shields et al., 2001,J Biol Chem 276:6591-6604; Shields et al., 2002, J Biol Chem277:26733-26740; Simmons et al., 2002, J Immunol Methods 263:133-147,incorporated by reference). Yet the carbohydrate makes little if anyspecific contact with FcγRs (Radaev et al., 2001, J Biol Chem276:16469-16477, incorporated by reference), indicating that thefunctional role of the N297 carbohydrate in mediating Fc/FcγR bindingmay be via the structural role it plays in determining the Fcconformation. This is supported by a collection of crystal structures offour different Fc glycoforms, which show that the composition of theoligosaccharide impacts the conformation of Cγ2 and as a result theFc/FcγR interface (Krapp et al., 2003, J Mol Biol 325:979-989,incorporated by reference).

The features of antibodies discussed above—specificity for target,ability to mediate immune effector mechanisms, and long half-life inserum—make antibodies powerful therapeutics. Monoclonal antibodies areused therapeutically for the treatment of a variety of conditionsincluding cancer, inflammation, and cardiovascular disease. There arecurrently over ten antibody products on the market and hundreds indevelopment. In addition to antibodies, an antibody-like protein that isfinding an expanding role in research and therapy is the Fc fusion(Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al.,1997, Curr Opin Immuno/9:195-200, incorporated by reference). An Fcfusion is a protein wherein one or more polypeptides is operably linkedto Fc. An Fc fusion combines the Fc region of an antibody, and thus itsfavorable effector functions and pharmacokinetics, with thetarget-binding region of a receptor, ligand, or some other protein orprotein domain. The role of the latter is to mediate target recognition,and thus it is functionally analogous to the antibody variable region.Because of the structural and functional overlap of Fc fusions withantibodies, the discussion on antibodies in the present inventionextends directly to Fc fusions.

There are a number of possible mechanisms by which antibodies destroytumor cells, including anti-proliferation via blockage of needed growthpathways, intracellular signaling leading to apoptosis, enhanced downregulation and/or turnover of receptors, CDC, ADCC, ADCP, and promotionof an adaptive immune response (Cragg et al., 1999, Curr Opin Immunol11:541-547; Glennie et al., 2000, Immunol Today 21:403-410. incorporatedby reference). Anti-tumor efficacy may be due to a combination of thesemechanisms, and their relative importance in clinical therapy appears tobe cancer dependent. Despite this arsenal of anti-tumor weapons, thepotency of antibodies as anti-cancer agents is unsatisfactory,particularly given their high cost. Patient tumor response data showthat monoclonal antibodies provide only a small improvement intherapeutic success over normal single-agent cytotoxicchemotherapeutics. For example, just half of all relapsed low-gradenon-Hodgkin's lymphoma patients respond to the anti-CD20 antibodyrituximab (McLaughlin et al., 1998, J Clin Oncol 16:2825-2833.incorporated by reference). Of 166 clinical patients, 6% showed acomplete response and 42% showed a partial response, with medianresponse duration of approximately 12 months. Trastuzumab (Herceptin®, aregistered trademark of Genentech), an anti-HER2/neu antibody fortreatment of metastatic breast cancer, has less efficacy. The overallresponse rate using trastuzumab for the 222 patients tested was only15%, with 8 complete and 26 partial responses and a median responseduration and survival of 9 to 13 months (Cobleigh et al., 1999, J ClinOnco/17:2639-2648, incorporated by reference). Currently for anticancertherapy, any small improvement in mortality rate defines success. Thusthere is a significant need to enhance the capacity of antibodies todestroy targeted cancer cells.

The role of FcγR-mediated effector functions in the anti-cancer activityof antibodies has been demonstrated in mice (Clynes et al., 1998, ProcNatl Acad Sci USA 95:652-656; Clynes et al., 2000, Nat Med 6:443-446,incorporated by reference), and the affinity of interaction between Fcand certain FcγRs correlates with targeted cytotoxicity in cell-basedassays (Shields et al., 2001, J Biol Chem 276:6591-6604; Presta et al.,2002, Biochem Soc Trans 30:487-490; Shields et al., 2002, J Biol Chem277:26733-26740, incorporated by reference). Additionally, a correlationhas been observed between clinical efficacy in humans and their allotypeof high (V158) or low (F158) affinity polymorphic forms of FcγRIIIa(Cartron et al., 2002, Blood 99:754-758, incorporated by reference).

Mutagenesis studies have been carried out on Fc towards various goals,with substitutions typically made to alanine (referred to as alaninescanning) or guided by sequence homology substitutions (Duncan et al.,1988, Nature 332:563-564; Lund et al., 1991, J Immuno/147:2657-2662;Lund et al., 1992, Mol Immunol 29:53-59; Jefferis et al., 1995, ImmunolLett 44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al.,1996, Immunol Lett 54:101-104; Lund et al., 1996, J Immunol157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Shieldset al., 2001, J Biol Chem 276:6591-6604) (U.S. Pat. No. 5,624,821; U.S.Pat. No. 5,885,573; PCT WO 00/42072; PCT WO 99/58572), all incorporatedby reference. The majority of substitutions reduce or ablate bindingwith FcγRs. However some success has been achieved at obtaining Fcvariants with higher FcγR affinity. (See for example U.S. Pat. No.5,624,821 and PCT WO 00/42072). For example, Winter and colleaguessubstituted the human amino acid at position 235 of mouse IgG2b antibody(a glutamic acid to leucine mutation) that increased binding of themouse antibody to human FcγRI by 100-fold (Duncan et al., 1988, Nature332:563-564) (U.S. Pat. No. 5,624,821). Shields et al. used alaninescanning mutagenesis to map Fc residues important to FcγR binding,followed by substitution of select residues with non-alanine mutations(Shields et al., 2001, J Biol Chem 276:6591-6604; Presta et al., 2002,Biochem Soc Trans 30:487-490) (PCT WO 00/42072), incorporated byreference.

Enhanced affinity of Fc for FcγR has also been achieved using engineeredglycoforms generated by expression of antibodies in engineered orvariant cell lines (Umañ a et al., 1999, Nat Biotechnol 17:176-180;Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002,J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem278:3466-3473, incorporated by reference). This approach has generatedenhancement of the capacity of antibodies to bind FcγRIIIa and tomediate ADCC.

Another major shortcoming of antibodies is their demanding productionrequirements (Garber, 2001, Nat Biotechnol 19:184-185; Dove, 2002, NatBiotechnol 20:777-779, incorporated by reference). Antibodies must beexpressed in mammalian cells, and the currently marketed antibodiestogether with other high-demand biotherapeutics consume essentially allof the available manufacturing capacity. With hundreds of biologics indevelopment, the majority of which are antibodies, there is an urgentneed for more efficient and cheaper methods of production. Thedownstream effects of insufficient antibody manufacturing capacity arethree-fold. First, it dramatically raises the cost of goods to theproducer, a cost that is passed on to the patient. Second, it hindersindustrial production of approved antibody products, limitingavailability of high demand therapeutics to patients. Finally, becauseclinical trials require large amounts of a protein that is not yetprofitable, the insufficient supply impedes progress of the growingantibody pipeline to market.

Alternative production methods have been explored in attempts atalleviating this problem. Transgenic plants and animals are beingpursued as potentially cheaper and higher capacity production systems(Chadd et al., 2001, Curr Opin Biotechnol 12:188-194, incorporated byreference). Such expression systems, however, can generate glycosylationpatterns significantly different from human glycoproteins. This mayresult in reduced or even lack of effector function because, asdiscussed above, the carbohydrate structure can significantly impactFcγR and complement binding. A potentially greater problem with nonhumanglycoforms may be immunogenicity; carbohydrates are a key source ofantigenicity for the immune system, and the presence of nonhumanglycoforms has a significant chance of eliciting antibodies thatneutralize the therapeutic, or worse cause adverse immune reactions.Thus the efficacy and safety of antibodies produced by transgenic plantsand animals remains uncertain. Bacterial expression is anotherattractive solution to the antibody production problem. Expression inbacteria, for example E. coli, provides a cost-effective and highcapacity method for producing proteins. For complex proteins such asantibodies there are a number of obstacles to bacterial expression,including folding and assembly of these complex molecules, properdisulfide formation, and solubility, stability, and functionality in theabsence of glycosylation because proteins expressed in bacteria are notglycosylated. Full length unglycosylated antibodies that bind antigenhave been successfully expressed in E. coli (Simmons et al., 2002, JImmunol Methods 263:133-147, incorporated by reference), and thus,folding, assembly, and proper disulfide formation of bacteriallyexpressed antibodies are possible in the absence of the eukaryoticchaperone machinery. However the ultimate utility of bacteriallyexpressed antibodies as therapeutics remains hindered by the lack ofglycosylation, which results in lack effector function and may result inpoor stability and solubility. This will likely be more problematic forformulation at the high concentrations for the prolonged periodsdemanded by clinical use.

In summary, there is a need for antibodies with enhanced therapeuticproperties.

SUMMARY OF THE INVENTION

The present invention provides Fc variants that are optimized for anumber of therapeutically relevant properties. These Fc variants aregenerally contained within a variant protein, that preferably comprisesan antibody or a Fc fusion protein.

It is an object of the present invention to provide novel Fc positionsat which amino acid modifications may be made to generate optimized Fcvariants. Said Fc positions include 230, 240, 244, 245, 247, 262, 263,266, 273, 275, 299, 302, 313, 323, 325, 328, and 332, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat. The present invention describes any amino acid modification atany of said novel Fc positions in order to generate an optimized Fcvariant.

It is a further object of the present invention to provide Fc variantsthat have been characterized herein. In one embodiment, said Fc variantscomprise at least one amino acid substitution at a position selectedfrom the group consisting of 221, 222, 223, 224, 225, 227, 228, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245,246, 247, 249, 255, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 282, 283, 284, 285,286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301,302, 303, 304, 305, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327,328, 329, 330, 331, 332, 333, 334, 335, 336, and 337, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat. In a preferred embodiment, said Fc variants comprise at least onesubstitution selected from the group consisting of D221K, D221Y, K222E,K222Y, T223E, T223K, H224E, H224Y, T225E, T225K, T225W, P227E, P227G,P227K, P227Y, P228E, P228G, P228K, P228Y, P230A, P230E, P230G, P230Y,A231E, A231G, A231K, A231P, A231Y, P232E, P232G, P232K, P232Y, E233A,E233D, E233F, E233G, E233H, E2331, E233K, E233L, E233M, E233N, E233Q,E233R, E233S, E233T, E233V, E233W, E233Y, L234A, L234D, L234E, L234F,L234G, L234H, L2341, L234K, L234M, L234N, L234P, L234Q, L234R, L2345,L234T, L234V, L234W, L234Y, L235A, L235D, L235E, L235F, L235G, L235H,L2351, L235K, L235M, L235N, L235P, L235Q, L235R, L2355, L235T, L235V,L235W, L235Y, G236A, G236D, G236E, G236F, G236H, G2361, G236K, G236L,G236M, G236N, G236P, G236Q, G236R, G236S, G236T, G236V, G236W, G236Y,G237D, G237E, G237F, G237H, G2371, G237K, G237L, G237M, G237N, G237P,G237Q, G237R, G237S, G237T, G237V, G237W, G237Y, P238D, P238E, P238F,P238G, P238H, P2381, P238K, P238L, P238M, P238N, P238Q, P238R, P238S,P238T, P238V, P238W, P238Y, S239D, S239E, S239F, S239G, S239H, S2391,S239K, S239L, S239M, S239N, S239P, S239Q, S239R, S239T, S239V, S239W,S239Y, V240A, V2401, V240M, V240T, F241D, F241E, F241L, F241R, F241S,F241W, F241Y, F243E, F243H, F243L, F243Q, F243R, F243W, F243Y, P244H,P245A, K246D, K246E, K246H, K246Y, P247G, P247V, D249H, D249Q, D249Y,R255E, R255Y, E258H, E258S, E258Y, T260D, T260E, T260H, T260Y, V262A,V262E, V262F, V2621, V262T, V263A, V2631, V263M, V263T, V264A, V264D,V264E, V264F, V264G, V264H, V2641, V264K, V264L, V264M, V264N, V264P,V264Q, V264R, V264S, V264T, V264W, V264Y, D265F, D265G, D265H, D2651,D265K, D265L, D265M, D265N, D265P, D265Q, D265R, D265S, D265T, D265V,D265W, D265Y, V266A, V2661, V266M, V266T, S267D, S267E, S267F, S267H,S2671, S267K, S267L, S267M, S267N, S267P, S267Q, S267R, S267T, S267V,S267W, S267Y, H268D, H268E, H268F, H268G, H2681, H268K, H268L, H268M,H268P, H268Q, H268R, H268T, H268V, H268W, E269F, E269G, E269H, E2691,E269K, E269L, E269M, E269N, E269P, E269R, E269S, E269T, E269V, E269W,E269Y, D270F, D270G, D270H, D2701, D270L, D270M, D270P, D270Q, D270R,D270S, D270T, D270W, D270Y, P271A, P271D, P271E, P271F, P271G, P271H,P2711, P271K, P271L, P271M, P271N, P271Q, P271R, P271S, P271T, P271V,P271W, P271Y, E272D, E272F, E272G, E272H, E2721, E272K, E272L, E272M,E272P, E272R, E272S, E272T, E272V, E272W, E272Y, V2731, K274D, K274E,K274F, K274G, K274H, K2741, K274L, K274M, K274N, K274P, K274R, K274T,K274V, K274W, K274Y, F275L, F275W, N276D, N276E, N276F, N276G, N276H,N2761, N276L, N276M, N276P, N276R, N276S, N276T, N276V, N276W, N276Y,Y278D, Y278E, Y278G, Y278H, Y2781, Y278K, Y278L, Y278M, Y278N, Y278P,Y278Q, Y278R, Y278S, Y278T, Y278V, Y278W, D280G, D280K, D280L, D280P,D280W, G281 D, G281 E, G281K, G281N, G281P, G281Q, G281Y, V282E, V282G,V282K, V282P, V282Y, E283G, E283H, E283K, E283L, E283P, E283R, E283Y,V284D, V284E, V284L, V284N, V284Q, V284T, V284Y, H285D, H285E, H285K,H285Q, H285W, H285Y, N286E, N286G, N286P, N286Y, K288D, K288E, K288Y,K290D, K290H, K290L, K290N, K290W, P291D, P291E, P291G, P291H, P2911,P291Q, P291T, R292D, R292E, R292T, R292Y, E293F, E293G, E293H, E2931,E293L, E293M, E293N, E293P, E293R, E293S, E293T, E293V, E293W, E293Y,E294F, E294G, E294H, E2941, E294K, E294L, E294M, E294P, E294R, E294S,E294T, E294V, E294W, E294Y, Q295D, Q295E, Q295F, Q295G, Q295H, Q2951,Q295M, Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, Y296A,Y296D, Y296E, Y296G, Y296H, Y2961, Y296K, Y296L, Y296M, Y296N, Y296Q,Y296R, Y296S, Y296T, Y296V, N297D, N297E, N297F, N297G, N297H, N2971,N297K, N297L, N297M, N297P, N297Q, N297R, N297S, N297T, N297V, N297W,N297Y, S298D, S298E, S298F, S298H, S2981, S298K, S298M, S298N, S298Q,S298R, S298T, S298W, S298Y, T299A, T299D, T299E, T299F, T299G, T299H,T2991, T299K, T299L, T299M, T299N, T299P, T299Q, T299R, T299S, T299V,T299W, T299Y, Y300A, Y300D, Y300E, Y300G, Y300H, Y300K, Y300M, Y300N,Y300P, Y300Q, Y300R, Y300S, Y300T, Y300V, Y300W, R301D, R301E, R301H,R301Y, V3021, V303D, V303E, V303Y, S304D, S304H, S304L, S304N, S304T,V305E, V305T, V305Y, W313F, K317E, K317Q, E318H, E318L, E318Q, E318R,E318Y, K320D, K320F, K320G, K320H, K3201, K320L, K320N, K320P, K320S,K320T, K320V, K320W, K320Y, K322D, K322F, K322G, K322H, K3221, K322P,K322S, K322T, K322V, K322W, K322Y, V3231, S324D, S324F, S324G, S324H,S3241, S324L, S324M, S324P, S324R, S324T, S324V, S324W, S324Y, N325A,N325D, N325E, N325F, N325G, N325H, N3251, N325K, N325L, N325M, N325P,N325Q, N325R, N325S, N325T, N325V, N325W, N325Y, K326I, K326L, K326P,K326T, A327D, A327E, A327F, A327H, A3271, A327K, A327L, A327M, A327N,A327P, A327R, A327S, A327T, A327V, A327W, A327Y, L328A, L328D, L328E,L328F, L328G, L328H, L3281, L328K, L328M, L328N, L328P, L328Q, L328R,L328S, L328T, L328V, L328W, L328Y, P329D, P329E, P329F, P329G, P329H,P3291, P329K, P329L, P329M, P329N, P329Q, P329R, P329S, P329T, P329V,P329W, P329Y, A330E, A330F, A330G, A330H, A3301, A330L, A330M, A330N,A330P, A330R, A330S, A330T, A330V, A330W, A330Y, P331D, P331F, P331H,P3311, P331L, P331M, P331Q, P331R, P331T, P331V, P331W, P331Y, I332A,I332D, I332E, I332F, I332H, I332K, I332L, I332M, I332N, I332P, I3320,I332R, I332S, I332T, I332V, I332W, I332Y, E333F, E333H, E3331, E333L,E333M, E333P, E333T, E333Y, K334F, K3341, K334L, K334P, K334T, T335D,T335F, T335G, T335H, T3351, T335L, T335M, T335N, T335P, T335R, T335S,T335V, T335W, T335Y, 1336E, 1336K, 1336Y, S337E, S337H, and S337N,wherein the numbering of the residues in the Fc region is that of the EUindex as in Kabat. This set of variants is sometimes referenced to as“the single variant set” of the invention.

It is an additional aspect of the invention to provide Fc variants (andproteins containing these variants) that have at least 1, 2, 3, 4, 5, 6,7, 8, 9 and 10 or more amino acid substitutions as compared to theparent Fc polypeptide, for example the Fc region SEQ ID NO:1. In someembodiments, 1, 2, 3 and 4 substitutions find particular use.

It is a further aspect of the invention to provide Fc variants (andproteins containing these variants) that exhibit altered Fc ligandbinding as compared to the parent Fc polypeptide, for example the Fcregion of SEQ ID NO:1, and that are encoded by nucleic acids thathybridize under high stringency conditions to a gene that encodes ahuman Fc polypeptide. High stringency conditions are known in the art;see for example U.S. Pat. No. 6,875,846, hereby incorporated byreference, particularly for high stringency conditions. Genes thatencode human Fc polypeptides are usually fragments of larger genes, andare also known in the art, as well as genes that due to the degeneracyof the genetic code will encode a naturally occurring Fc polypeptideeven if not naturally occurring themselves.

It is an additional aspect of the invention to provide Fc variants (andproteins containing these variants) that have at least 1, 2, 3, 4, 5, 6,7, 8, 9 and 10 or more amino acid substitutions as compared to theparent Fc polypeptide, for example the Fc region SEQ ID NO:1. In someembodiments, 1, 2, 3 and 4 substitutions find particular use.

It is a further aspect of the invention to provide Fc variants (andproteins containing these variants) that exhibit altered Fc ligandbinding as compared to the parent Fc polypeptide, for example the Fcregion of SEQ ID NO:1, and that are encoded by nucleic acids thathybridize under high stringency conditions to a gene that encodes ahuman Fc polypeptide. High stringency conditions are known in the art;see for example U.S. Pat. No. 6,875,846, hereby incorporated byreference, particularly for high stringency conditions. Genes thatencode human Fc polypeptides are usually fragments of larger genes, andare also known in the art, as well as genes that due to the degeneracyof the genetic code will encode a naturally occurring Fc polypeptideeven if not naturally occurring themselves.

It is an additional aspect of the invention to provide for variant Fcpolypeptides that exhibit altered ADCC activity, particularly increasedADCC activity. In some aspects, these variants comprise an amino acidsubstitution at position 239, optionally amino acid substitutions atpositions 239 and 332, and optionally can include any othersubstitutions outlined in the single variant set above, to createvariants comprising multiple substitutions. It is a further object ofthe present invention to provide Fc variants that have beencharacterized herein, wherein said Fc variants are selected from thegroup consisting of D221K, D221Y, K222E, K222Y, T223E, T223K, H224E,H224Y, T225E, T225K, T225W, P227E, P227G, P227K, P227Y, P228E, P228G,P228K, P228Y, P230A, P230A/E233D, P230A/E233D/I332E, P230E, P230G,P230Y, A231E, A231G, A231K, A231P, A231Y, P232E, P232G, P232K, P232Y,E233A, E233D, E233F, E233G, E233H, E2331, E233K, E233L, E233M, E233N,E233Q, E233R, E233S, E233T, E233V, E233W, E233Y, L234A, L234D, L234E,L234F, L234G, L234H, L234I, L234I, L235D, L234K, L234M, L234N, L234P,L234Q, L234R, L2345, L234T, L234V, L234W, L234Y, L235A, L235D,L235D/S239D/A330Y/I332E, L235D/S239D/N297D/I332E, L235E, L235F, L235G,L235H, L235I, L235K, L235M, L235N, L235P, L235Q, L235R, L2355, L235T,L235V, L235W, L235Y, G236A, G236D, G236E, G236F, G236H, G2361, G236K,G236L, G236M, G236N, G236P, G236Q, G236R, G236S, G236T, G236V, G236W,G236Y, G237D, G237E, G237F, G237H, G2371, G237K, G237L, G237M, G237N,G237P, G237Q, G237R, G237S, G237T, G237V, G237W, G237Y, P238D, P238E,P238F, P238G, P238H, P2381, P238K, P238L, P238M, P238N, P238Q, P238R,P238S, P238T, P238V, P238W, P238Y, S239D, S239D/A330L/I332E,S239D/A330Y/I332E/L234I, S239D/A330Y/I332E/V266I,S239D/D265F/N297D/I332E, S239D/D265H/N297D/I332E,S239D/D2651/N297D/I332E, S239D/D265L/N297D/I332E,S239D/D265T/N297D/I332E, S239D/D265Y/N297D/I332E,S239D/E2721/A330L/I332E, S239D/E272I/I332E, S239D/E272K/A330L/I332E,S239D/E272K/I332E, S239D/E272S/A330L/I332E, S239D/E272S/I332E,S239D/E272Y/A330L/I332E, S239D/E272Y/I332E,S239D/F241S/F243H/V262T/V264T/N297D/A330Y/I332E, S239D/H268D,S239D/H268E, S239D/I332D, S239D/I332E, S239D/I332E/A327D,S239D/I332E/A3301, S239D/I332E/A330Y, S239D/I332E/E272H,S239D/I332E/E272R, S239D/I332E/E283H, S239D/I332E/E283L,S239D/I332E/G236A, S239D/I332E/G236S, S239D/I332E/H268D,S239D/I332E/H268E, S239D/I332E/K246H, S239D/I332E/R255Y,S239D/I332E/S267E, S239D/I332E/V2641, S239D/I332E/V2641/A330L,S239D/I332E/V2641/S298A, S239D/I332E/V284D, S239D/I332E/V284E,S239D/I332E/V284E, S239D/I332N, S239D/I332Q, S239D/K274E/A330L/I332E,S239D/K274E/I332E, S239D/K326E/A330L/I332E, S239D/K326E/A330Y/I332E,S239D/K326E/I332E, S239D/K326T/A330Y/I332E, S239D/K326T/I332E,S239D/N297D/A330Y/I332E, S239D/N297D/I332E, S239D/N297D/K326E/I332E,S239D/S267E/A330L/I332E, S239D/S267E/I332E, S239D/S298A/K326E/I332E,S239D/S298A/K326T/I332E, S239D/V2401/A330Y/I332E,S239D/V264T/A330Y/I332E, S239D/Y278T/A330L/I332E, S239D/Y278T/I332E,S239E, S239E/D265G, S239E/D265N, S239E/D265Q, S239E/I332D, S239E/I332E,S239E/I332N, S239E/I332Q, S239E/N297D/I332E, S239E/V264I/A330Y/I332E,S239E/V264I/I332E, S239E/V2641/S298A/A330Y/I332E, S239F, S239G, S239H,S2391, S239K, S239L, S239M, S239N, S239N/I332D, S239N/I332E,S239N/I332E/A330L, S239N/I332E/A330Y, S239N/I332N, S239N/I332Q, S239P,S239Q, S239Q/I332D, S239Q/I332E, S239Q/I332N, S239Q/I332Q,S239Q/V264I/I332E, S239R, S239T, S239V, S239W, S239Y, V240A, V2401,V240I/V2661, V240M, V240T, F241 D, F241 E,F241E/F243Q/V262T/V264E/I332E, F241E/F243Q/V262T/V264E,F241E/F243R/V262E/V264R/I332E, F241E/F243R/V262E/V264R,F241E/F243Y/V262T/V264R/I332E, F241E/F243Y/V262T/V264R, F241L,F241L/F243L/V262I/V264I, F241L/V262I, F241R/F243Q/V262T/V264R/I332E,F241R/F243Q/V262T/V264R, F241 W, F241 W/F243W, F241W/F243W/V262A/V264A,F241 Y, F241Y/F243Y/V262T/V264T/N297D/I332E, F241Y/F243Y/V262T/V264T,F243E, F243L, F243L/V262I/V264W, F243L/V264I, F243W, P244H,P244H/P245A/P247V, P245A, K246D, K246E, K246H, K246Y, P247G, P247V,D249H, D249Q, D249Y, R255E, R255Y, E258H, E258S, E258Y, T260D, T260E,T260H, T260Y, V262E, V262F, V263A, V2631, V263M, V263T, V264A, V264D,V264E, V264E/N297D/I332E, V264F, V264G, V264H, V2641, V2641/A330L/I332E,V2641/A330Y/I332E, V264I/I332E, V264K, V264L, V264M, V264N, V264P,V264Q, V264R, V264S, V264T, V264W, V264Y, D265F, D265F/N297E/I332E,D265G, D265H, D2651, D265K, D265L, D265M, D265N, D265P, D265Q, D265R,D265S, D265T, D265V, D265W, D265Y, D265Y/N297D/I332E,D265Y/N297D/T299L/I332E, V266A, V2661, V266M, V266T, S267D, S267E,S267E, S267E/A327D, S267E/P331D, S267E/S324I, S267E/V282G, S267F, S267H,S2671, S267K, S267L, S267L/A327S, S267M, S267N, S267P, S267Q,S267Q/A327S, S267R, S267T, S267V, S267W, S267Y, H268D, H268E, H268F,H268G, H2681, H268K, H268L, H268M, H268P, H268Q, H268R, H268T, H268V,H268W, E269F, E269G, E269H, E2691, E269K, E269L, E269M, E269N, E269P,E269R, E269S, E269T, E269V, E269W, E269Y, D270F, D270G, D270H, D2701,D270L, D270M, D270P, D270Q, D270R, D270S, D270T, D270W, D270Y, P271A,P271D, P271E, P271F, P271G, P271H, P2711, P271K, P271L, P271M, P271N,P271Q, P271R, P271S, P271T, P271V, P271W, P271Y, E272D, E272F, E272G,E272H, E2721, E272K, E272L, E272M, E272P, E272R, E272S, E272T, E272V,E272W, E272Y, V2731, K274D, K274E, K274F, K274G, K274H, K274I, K274L,K274M, K274N, K274P, K274R, K274T, K274V, K274W, K274Y, F275L, F275W,N276D, N276E, N276F, N276G, N276H, N2761, N276L, N276M, N276P, N276R,N276S, N276T, N276V, N276W, N276Y, Y278D, Y278E, Y278G, Y278H, Y2781,Y278K, Y278L, Y278M, Y278N, Y278P, Y278Q, Y278R, Y278S, Y278T, Y278V,Y278W, Y278W, Y278W/E283R/V3021, Y278W/V3021, D280G, D280K, D280L,D280P, D280W, G281D, G281D/V282G, G281E, G281K, G281N, G281P, G281Q,G281Y, V282E, V282G, V282G/P331D, V282K, V282P, V282Y, E283G, E283H,E283K, E283L, E283P, E283R, E283R/V302I/Y278W/E283R, E283Y, V284D,V284E, V284L, V284N, V284Q, V284T, V284Y, H285D, H285E, H285K, H285Q,H285W, H285Y, N286E, N286G, N286P, N286Y, K288D, K288E, K288Y, K290D,K290H, K290L, K290N, K290W, P291D, P291E, P291G, P291H, P2911, P291Q,P291T, R292D, R292E, R292T, R292Y, E293F, E293G, E293H, E2931, E293L,E293M, E293N, E293P, E293R, E293S, E293T, E293V, E293W, E293Y, E294F,E294G, E294H, E2941, E294K, E294L, E294M, E294P, E294R, E294S, E294T,E294V, E294W, E294Y, Q295D, Q295E, Q295F, Q295G, Q295H, O295I, Q295M,Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, Y296A, Y296D,Y296E, Y296G, Y2961, Y296K, Y296L, Y296M, Y296N, Y296Q, Y296R, Y296S,Y296T, Y296V, N297D, N297D/I332E, N297D/I332E/A330Y,N297D/I332E/S239D/A330L, N297D/I332E/S239D/D265V,N297D/I332E/S298A/A330Y, N297D/I332E/T299E, N297D/I332E/T299F,N297D/I332E/T299H, N297D/I332E/T2991, N297D/I332E/T299L,N297D/I332E/T299V, N297D/I332E/Y296D, N297D/I332E/Y296E,N297D/I332E/Y296H, N297D/I332E/Y296N, N297D/I332E/Y296Q,N297D/I332E/Y296T, N297E/I332E, N297F, N297G, N297H, N2971, N297K,N297L, N297M, N297P, N297Q, N297R, N297S, N297S/I332E, N297T, N297V,N297W, N297Y, S298A/I332E, S298A/K326E, S298A/K326E/K334L, S298A/K334L,S298D, S298E, S298F, S298H, S2981, S298K, S298M, S298N, S298Q, S298R,S298T, S298W, S298Y, T299A, T299D, T299E, T299F, T299G, T299H, T2991,T299K, T299L, T299M, T299N, T299P, T299Q, T299R, T299S, T299V, T299W,T299Y, Y300A, Y300D, Y300E, Y300G, Y300H, Y300K, Y300M, Y300N, Y300P,Y300Q, Y300R, Y300S, Y300T, Y300V, Y300W, R301D, R301E, R301H, R301Y,V302I, V303D, V303E, V303Y, S304D, S304H, S304L, S304N, S304T, V305E,V305T, V305Y, W313F, K317E, K317Q, E318H, E318L, E318Q, E318R, E318Y,K320D, K320F, K320G, K320H, K3201, K320L, K320N, K320P, K320S, K320T,K320V, K320W, K320Y, K322D, K322F, K322G, K322H, K3221, K322P, K322S,K322T, K322V, K322W, K322Y, V3231, S324D, S324F, S324G, S324H, S3241,S324I/A327D, S324L, S324M, S324P, S324R, S324T, S324V, S324W, S324Y,N325A, N325D, N325E, N325F, N325G, N325H, N3251, N325K, N325L, N325M,N325P, N325Q, N325R, N325S, N325T, N325V, N325W, N325Y, K326I, K326L,K326P, K326T, A327D, A327E, A327F, A327H, A3271, A327K, A327L, A327M,A327N, A327P, A327R, A327S, A327T, A327V, A327W, A327Y, L328A, L328D,L328D/I332E, L328E, L328E/I332E, L328F, L328G, L328H, L328H/I332E,L3281, L328I/I332E, L328I/I332E, L328K, L328M, L328M/I332E, L328N,L328N/I332E, L328P, L328Q, L328Q/I332E, L328Q/I332E, L328R, L328S,L328T, L328T/I332E, L328V, L328V/I332E, L328W, L328Y, P329D, P329E,P329F, P329G, P329H, P3291, P329K, P329L, P329M, P329N, P329Q, P329R,P329S, P329T, P329V, P329W, P329Y, A330E, A330F, A330G, A330H, A3301,A330L, A330L/I332E, A330M, A330N, A330P, A330R, A330S, A330T, A330V,A330W, A330Y, A330Y/I332E, P331D, P331F, P331H, P331I, P331L, P331M,P331Q, P331R, P331T, P331V, P331W, P331Y, I332A, I332D, I332E,I332E/G281D, I332E/H268D, I332E/H268E, I332E/S239D/S298A,I332E/S239N/S298A, I332E/V264I/S298A, I332E/V284E, I332F, I332H, I332K,I332L, I332M, I332N, I332P, I332Q, I332R, I332S, I332T, I332V, I332W,I332Y, E333F, E333H, E3331, E333L, E333M, E333P, E333T, E333Y, K334F,K3341, K334P, K334T, T335D, T335F, T335G, T335H, T3351, T335L, T335M,T335N, T335P, T335R, T335S, T335V, T335W, T335Y, 1336E, I336K, 1336Y,S337E, S337H, and S337N, wherein the numbering of the residues in the Fcregion is that of the EU index as in Kabat.

It is a further object of the present invention to provide an Fc variantthat binds with greater affinity to one or more FcγRs. In oneembodiment, said Fc variants have affinity for an FcγR that is more than1-fold greater than that of the parent Fc polypeptide. In an alternateembodiment, said Fc variants have affinity for an FcγR that is more than5-fold greater than that of the parent Fc polypeptide. In a preferredembodiment, said Fc variants have affinity for an FcγR that is betweenabout 5-fold and 300-fold greater than that of the parent Fcpolypeptide.

It is a further object of the present invention to provide Fc variantthat have a FcγRIIIa-fold:FcγRIIb-fold ratio greater than 1:1. In oneembodiment, said Fc variants have a FcγRIIIa-fold:FcγRIIb-fold ratiogreater than 11:1. In a preferred embodiment, said Fc variants have aFcγRIIIa-fold:FcγRIIb-fold ratio between 11:1 and 86:1.

It is a further object of the present invention to provide Fc variantsthat mediate effector function more effectively in the presence ofeffector cells. In one embodiment, said Fc variants mediate ADCC that isgreater than that mediated by the parent Fc polypeptide. In a preferredembodiment, said Fc variants mediate ADCC that is more than 5-foldgreater than that mediated by the parent Fc polypeptide. In a mostlypreferred embodiment, said Fc variants mediate ADCC that is between5-fold and 1000-fold greater than that mediated by the parent Fcpolypeptide.

It is a further object of the present invention to provide Fc variantsthat bind with weaker affinity to one or more FcγRs. It is a furtherobject of the present invention to provide Fc variants that mediate ADCCin the presence of effector cells less effectively.

It is a further object of the present invention to provide Fc variantsthat have improved function and/or solution properties as compared tothe aglycosylated form of the parent Fc polypeptide. Improvedfunctionality herein includes but is not limited to binding affinity toan Fc ligand. Improved solution properties herein includes but is notlimited to stability and solubility. In an one embodiment, said Fcvariants bind to an FcγR with an affinity that is within about 0.5-foldof the glycosylated form of the parent Fc polypeptide. In an alternateembodiment, said aglycosylated Fc variants bind to an FcγR with anaffinity that is comparable to the glycosylated parent Fc polypeptide.In an alternate embodiment, said Fc variants bind to an FcγR with anaffinity that is greater than the glycosylated form of the parent Fcpolypeptide.

The present invention also provides methods for engineering optimized Fcvariants. It is a further object of the present invention to provideexperimental production and screening methods for obtaining optimized Fcvariants.

The present invention provides isolated nucleic acids encoding the Fcvariants described herein. The present invention provides vectorscomprising said nucleic acids, optionally, operably linked to controlsequences. The present invention provides host cells containing thevectors, and methods for producing and optionally recovering the Fcvariants.

The present invention provides novel Fc polypeptides, includingantibodies, Fc fusions, isolated Fc, and Fc fragments, that comprise theFc variants disclosed herein. Said novel Fc polypeptides may find use ina therapeutic product.

The present invention provides compositions comprising Fc polypeptidesthat comprise the Fc variants described herein, and a physiologically orpharmaceutically acceptable carrier or diluent.

The present invention contemplates therapeutic and diagnostic uses forFc polypeptides that comprise the Fc variants disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Antibody structure and function. Shown is a model of a fulllength human IgG1 antibody, modeled using a humanized Fab structure frompdb accession code 10E1 (James et al., 1999, J Mol Biol 289:293-301) anda human IgG1 Fc structure from pdb accession code 1DN2 (DeLano et al.,2000, Science 287:1279-1283). The flexible hinge that links the Fab andFc regions is not shown. IgG1 is a homodimer of heterodimers, made up oftwo light chains and two heavy chains. The Ig domains that comprise theantibody are labeled, and include V_(L) and C_(L) for the light chain,and V_(H), Cgamma1 (Cγ1), Cgamma2 (Cγ2), and Cgamma3 (Cγ3) for the heavychain. The Fc region is labeled. Binding sites for relevant proteins arelabeled, including the antigen binding site in the variable region, andthe binding sites for FcγRs, FcRn, C1q, and proteins A and G in the Fcregion.

FIG. 2. The Fc/FcγRIIIb complex structure 1IIS. Fc is shown as a grayribbon diagram, and FcγRIIIb is shown as a black ribbon. The N297carbohydrate is shown as black sticks.

FIGS. 3 a-3 b. Alignment of the amino acid sequences of the human IgGimmunoglobulins IgG1, IgG2, IgG3, and IgG4 (SEQ ID NOs. 1-4]. FIG. 3 aprovides the sequences of the CH1 (Cγ1) and hinge domains, and FIG. 3 bprovides the sequences of the CH2 (Cγ2) and CH3 (Cγ3) domains. Positionsare numbered according to the EU index of the IgG1 sequence, anddifferences between IgG1 and the other immunoglobulins IgG2, IgG3, andIgG4 are shown in grey. Polymorphisms exist at a number of positions(Kim et al., 2001, J. Mol. Evol. 54:1-9), and thus slight differencesbetween the presented sequences and sequences in the prior art mayexist. The possible beginnings of the Fc region are labeled, definedherein as either EU position 226 or 230.

FIG. 4. Residues at which amino acid modifications were made in the Fcvariants of the present invention, mapped onto the Fc/FcγRIIIb complexstructure 1IIS. Fc is shown as a gray ribbon diagram, and FcγRIIIb isshown as a black ribbon. Experimental library residues are shown inblack, the N297 carbohydrate is shown in grey.

FIG. 5. Expression of Fc variant and wild type (WT) proteins ofalemtuzumab in 293T cells. Plasmids containing alemtuzumab heavy chaingenes (WT or variants) were co-transfected with plasmid containing thealemtuzumab light chain gene. Media were harvested 5 days aftertransfection. For each transfected sample, 10 ul medium was loaded on aSDS-PAGE gel for Western analysis. The probe for Western wasperoxidase-conjugated goat-anti human IgG (Jackson Immuno-Research,catalog #109-035-088). WT: wild type alemtuzumab; 1-10: alemtuzumabvariants. H and L indicate antibody heavy chain and light chain,respectively.

FIG. 6. Purification of alemtuzumab using protein A chromatography. WTalemtuzumab proteins was expressed in 293T cells and the media washarvested 5 days after transfection. The media were diluted 1:1 with PBSand purified with protein A (Pierce, Catalog #20334). O: original samplebefore purification; FT: flow through; E: elution; C: concentrated finalsample. The left picture shows a Simple Blue-stained SDS-PAGE gel, andthe right shows a western blot labeled using peroxidase-conjugatedgoat-anti human IgG.

FIG. 7. Production of deglycosylated antibodies. Wild type and variantsof alemtuzumab were expressed in 293T cells and purified with protein Achromatography. Antibodies were incubated with peptide-N-glycosidase(PNGase F) at 37° C. for 24 h. For each antibody, a mock treated sample(-PNGase F) was done in parallel. WT: wild-type alemtuzumab; #15, #16,#17, #18, #22: alemtuzumab variants F241E/F243R/V262E/V264R,F241E/F243Q/V262T/V264E, F241R/F243Q/V262T/V264R,F241E/F243Y/V262T/V264R, and I332E respectively. The faster migration ofthe PNGase F treated versus the mock treated samples represents thedeglycosylated heavy chains.

FIG. 8. Alemtuzumab expressed from 293T cells binds its antigen. Theantigenic CD52 peptide, fused to GST, was expressed in E. coli BL21(DE3) under IPTG induction. Both uninduced and induced samples were runon a SDS-PAGE gel, and transferred to PVDF membrane. For westernanalysis, either alemtuzumab from Sotec (a-CD52, Sotec) (finalconcentration 2.5 ng/ul) or media of transfected 293T cells (Campath,Xencor) (final alemtuzumab concentration approximately 0.1-0.2 ng/ul)were used as primary antibody, and peroxidase-conjugated goat-anti humanIgG was used as secondary antibody. M: pre-stained marker; U: un-inducedsample for GST-CD52; 1: induced sample for GST-CD52.

FIG. 9. Expression and purification of extracellular region of humanV158 FcγRIIIa. Tagged FcγRIIIa was transfected in 293T cells, and mediacontaining secreted FcγRIIIa were harvested 3 days later and purifiedusing affinity chromatography. 1: media; 2: flow through; 3: wash; 4-8:serial elutions. Both simple blue-stained SDS-PAGE gel and westernresult are shown. For the western blot, membrane was probed withanti-GST antibody.

FIG. 10. Binding to human V158 FcγRIIIa by select alemtuzumab Fcvariants from the experimental library as determined by the AlphaScreen™assay, described in Example 2. In the presence of competitor antibody(Fc variant or WT alemtuzumab) a characteristic inhibition curve isobserved as a decrease in luminescence signal. Phosphate buffer saline(PBS) alone was used as the negative control. The binding data werenormalized to the maximum and minimum luminescence signal for eachparticular curve, provided by the baselines at low and high antibodyconcentrations respectively. The curves represent the fits of the datato a one site competition model using nonlinear regression. These fitsprovide IC50s for each antibody, illustrated for WT and S239D by thedotted lines.

FIGS. 11 a and 11 b. AlphaScreen assay showing binding of selectalemtuzumab (FIG. 11 a) and trastuzumab (FIG. 11 b) Fc variants to humanVal158 FcγRIIIa. The binding data were normalized to the upper and lowerbaselines for each particular antibody, and the curves represent thefits of the data to a one site competition model. PBS was used as anegative control.

FIG. 12. AlphaScreen assay showing binding of select alemtuzumab Fcvariants to human FcγRIIb. The binding data were normalized to the upperand lower baselines for each particular antibody, and the curvesrepresent the fits of the data to a one site competition model. PBS wasused as a negative control.

FIG. 13. AlphaScreen assay showing binding of select alemtuzumab Fcvariants to human R131FcγRIIa. The binding data were normalized to theupper and lower baselines for each particular antibody, and the curvesrepresent the fits of the data to a one site competition model.

FIG. 14. AlphaScreen assay measuring binding of select alemtuzumab Fcvariants to human FcRn, as described in Example 2. The binding data werenormalized to the upper and lower baselines for each particularantibody, and the curves represent the fits of the data to a one sitecompetition model. PBS was used as a negative control.

FIG. 15. AlphaScreen assay measuring binding of select alemtuzumab Fcvariants to bacterial protein A, as described in Example 2. The bindingdata were normalized to the upper and lower baselines for eachparticular antibody, and the curves represent the fits of the data to aone site competition model. PBS was used as a negative control.

FIGS. 16 a-16 b. AlphaScreen assay comparing binding of selectalemtuzumab Fc variants to human V158 FcγRIIIa (FIG. 16 a) and humanFcγRIIb (FIG. 16 b). The binding data were normalized to the upper andlower baselines for each particular antibody, and the curves representthe fits of the data to a one site competition model. PBS was used as anegative control.

FIGS. 17 a-17 c. AlphaScreen assay measuring binding to human V158FcγRIIIa (FIGS. 17 a and 17 b) and human FcγRIIb (FIG. 17 c) by selectFc variants in the context of trastuzumab. The binding data werenormalized to the upper and lower baselines for each particularantibody, and the curves represent the fits of the data to a one sitecompetition model. PBS was used as a negative control.

FIG. 18. AlphaScreen assay measuring binding to human V158 FcγRIIIa byselect Fc variants in the context of rituximab. The binding data werenormalized to the upper and lower baselines for each particularantibody, and the curves represent the fits of the data to a one sitecompetition model. PBS was used as a negative control.

FIG. 19. AlphaScreen assay measuring binding to human V158 FcγRIIIa byselect Fc variants in the context of cetuximab. The binding data werenormalized to the upper and lower baselines for each particularantibody, and the curves represent the fits of the data to a one sitecompetition model. PBS was used as a negative control.

FIGS. 20 a-20 b. AlphaScreen assay showing binding of select alemtuzumabFc variants to the V158 (FIG. 20 a) and F158 (FIG. 20 b) allotypes ofhuman FcγRIIIa. The binding data were normalized to the upper and lowerbaselines for each particular antibody, and the curves represent thefits of the data to a one site competition model. PBS was used as anegative control.

FIGS. 21 a-21 d. FIGS. 21 a and 21 b show the correlation between SPRKd's and AlphaScreen IC50's from binding of select alemtuzumab Fcvariants to V158 FcγRIIIa (FIG. 21 a) and F158 FcγRIIIa (FIG. 21 b).FIGS. 21 c and 21 d show the correlation between SPR and AlphaScreenfold-improvements over WT for binding of select alemtuzumab Fc variantsto V158 FcγRIIIa (FIG. 21 c) and F158 FcγRIIIa (FIG. 21 d). Binding dataare presented in Table 3. The lines through the data represent thelinear fits of the data, and the r² values indicate the significance ofthese fits.

FIGS. 22 a and 22 b. AlphaScreen assay showing binding of selectalemtuzumab Fc variants to human V158 FcγRIIIa. The binding data werenormalized to the upper and lower baselines for each particularantibody, and the curves represent the fits of the data to a one sitecompetition model. PBS was used as a negative control.

FIGS. 23 a-23 b. Cell-based ADCC assays of select Fc variants in thecontext of alemtuzumab. ADCC was measured using the DELFIA® EuTDA-basedcytotoxicity assay (Perkin Elmer, Mass.), as described in Example 3,using DoHH-2 lymphoma target cells and 50-fold excess human PBMCs. FIG.23 a is a bar graph showing the raw fluorescence data for the indicatedalemtuzumab antibodies at 10 ng/ml. The PBMC bar indicates basal levelsof cytotoxicity in the absence of antibody. FIG. 23 b shows thedose-dependence of ADCC on antibody concentration for the indicatedalemtuzumab antibodies, normalized to the minimum and maximumfluorescence signal for each particular curve, provided by the baselinesat low and high antibody concentrations respectively. The curvesrepresent the fits of the data to a sigmoidal dose-response model usingnonlinear regression.

FIGS. 24 a-24 c. Cell-based ADCC assays of select Fc variants in thecontext of trastuzumab. ADCC was measured using the DELFIA® EuTDA-basedcytotoxicity assay, as described in Example 3, using BT474 and Sk-Br-3breast carcinoma target cells and 50-fold excess human PBMCs. FIG. 24 ais a bar graph showing the raw fluorescence data for the indicatedtrastuzumab antibodies at 1 ng/ml. The PBMC bar indicates basal levelsof cytotoxicity in the absence of antibody. FIGS. 24 b and 24 c show thedose-dependence of ADCC on antibody concentration for the indicatedtrastuzumab antibodies, normalized to the minimum and maximumfluorescence signal for each particular curve, provided by the baselinesat low and high antibody concentrations respectively. The curvesrepresent the fits of the data to a sigmoidal dose-response model usingnonlinear regression.

FIGS. 25 a-25 c. Cell-based ADCC assays of select Fc variants in thecontext of rituximab. ADCC was measured using the DELFIA® EuTDA-basedcytotoxicity assay, as described in Example 3, using WIL2-S lymphomatarget cells and 50-fold excess human PBMCs. FIG. 25 a is a bar graphshowing the raw fluorescence data for the indicated rituximab antibodiesat 1 ng/ml. The PBMC bar indicates basal levels of cytotoxicity in theabsence of antibody. FIGS. 25 b and 25 c show the dose-dependence ofADCC on antibody concentration for the indicated rituximab antibodies,normalized to the minimum and maximum fluorescence signal for eachparticular curve, provided by the baselines at low and high antibodyconcentrations respectively. The curves represent the fits of the datato a sigmoidal dose-response model using nonlinear regression.

FIGS. 26 a-26 b. Cell-based ADCC assay of select trastuzumab (FIG. 26 a)and rituximab (FIG. 26 b) Fc variants showing enhancements in potencyand efficacy. Both assays used homozygous F158/F158 FcγRIIIa PBMCs aseffector cells at a 25-fold excess to target cells, which were Sk-Br-3for the trastuzumab assay and WIL2-S for the rituximab assay. Data werenormalized according to the absolute minimal lysis for the assay,provided by the fluorescence signal of target cells in the presence ofPBMCs alone (no antibody), and the absolute maximal lysis for the assay,provided by the fluorescence signal of target cells in the presence ofTriton X1000, as described in Example 3.

FIG. 27. AlphaScreen assay showing binding of select alemtuzumab Fcvariants to human V158 FcγRIIIa. The binding data were normalized to theupper and lower baselines for each particular antibody, and the curvesrepresent the fits of the data to a one site competition model. PBS wasused as a negative control.

FIG. 28. ADCC. Cell-based ADCC assays of select Fc variant trastuzumabantibodies as compared to WT trastuzumab. Purified human peripheralblood monocytes (PBMCs) were used as effector cells, and Sk-Br-3 breastcarcinoma cells were used as target cells. Lysis was monitored bymeasuring LDH activity using the Cytotoxicity Detection Kit (LDH, RocheDiagnostic Corporation, Indianapolis, Ind.). Samples were run intriplicate to provide error estimates (n=3, +/− S.D.). The figure showsthe dose dependence of ADCC at various antibody concentrations,normalized to the minimum and maximum levels of lysis for the assay. Thecurves represent the fits of the data to a sigmoidal dose-response modelusing nonlinear regression.

FIGS. 29 a-29 b. Cell-based ADCC assay of select trastuzumab Fc variantsagainst different cell lines expressing varying levels of the Her2/neutarget antigen. ADCC assays were run as described in Example 5, withvarious cell lines expressing amplified to low levels of Her2/neureceptor, including Sk-Br-3 (1×106 copies), SkOV3 (˜1×105),OVCAR3(˜1×104), and MCF-7 (˜3×103 copies). FIG. 29 a provides a westernblot showing the Her2 expression level for each cell line; equivalentamounts of cell lysate were loaded on an SDS-PAGE gel, and Her2 wasdetected using trastuzumab. Human PBMCs allotyped as homozygousF158/F158 FcγRIIIa were used at 25-fold excess to target cells. The bargraph in FIG. 29 b provides ADCC data for WT and Fc variant against theindicated cell lines, normalized to the minimum and maximum fluorescencesignal provided by minimal lysis (PBMCs alone) and maximal lysis (TritonX1000).

FIG. 30. Cell-based ADCC assays of select Fc variants in the context oftrastuzumab using natural killer (NK) cells as effector cells andmeasuring LDH release to monitor cell lysis. NK cells, allotyped asheterozygous F158/F158 FcγRIIIa, were at an 4-fold excess to Sk-Br-3breast carcinoma target cells, and the level of cytotoxicity wasmeasured using the LDH Cytotoxicity Detection Kit, according to themanufacturer's protocol (Roche Diagnostics GmbH, Penzberg, Germany). Thegraph shows the dose-dependence of ADCC on antibody concentration forthe indicated trastuzumab antibodies, normalized to the minimum andmaximum fluorescence signal for each particular curve, provided by thebaselines at low and high antibody concentrations respectively. Thecurves represent the fits of the data to a sigmoidal dose-response modelusing nonlinear regression.

FIG. 31. Cell-based ADCP assay of select variants. The ADCP assay wascarried out as described in Example 7, using a co-labeling strategycoupled with flow cytometry. Differentiated macrophages were used aseffector cells, and Sk-Br-3 cells were used as target cells. Percentphagocytosis represents the number of co-labeled cells(macrophage+Sk-Br-3) over the total number of Sk-Br-3 in the population(phagocytosed+non-phagocytosed).

FIGS. 32 a-32 c. Capacity of select Fc variants to mediate binding andactivation of complement. FIG. 32 a shows an AlphaScreen assay measuringbinding of select alemtuzumab Fc variants to C1q. The binding data werenormalized to the upper and lower baselines for each particularantibody, and the curves represent the fits of the data to a one sitecompetition model. FIGS. 32 b and 31 c show a cell-based assay measuringcapacity of select rituximab Fc variants to mediate CDC. CDC assays wereperformed using Alamar Blue to monitor lysis of Fc variant and WTrituximab-opsonized WIL2-S lymphoma cells by human serum complement(Quidel, San Diego, Calif.). The dose-dependence on antibodyconcentration of complement-mediated lysis is shown for the indicatedrituximab antibodies, normalized to the minimum and maximum fluorescencesignal for each particular curve, provided by the baselines at low andhigh antibody concentrations respectively. The curves represent the fitsof the data to a sigmoidal dose-response model using nonlinearregression.

FIGS. 33 a-33 c. Enhanced B cell depletion by Fc variants in macaques,as described in Example 9. FIG. 33 a shows the percent B cells remainingin Macaca Fascicularis monkeys during treatment with anti-CD20 WT andS239D/I332E rituximab antibodies, measured using markers CD20+ andCD40+. FIG. 33 b shows the percent natural killer (NK) cells remainingin the monkeys during treatment, measured using markers CD3−/CD16+ andCD3−/CD8+. FIG. 33 c shows the dose response of CD20+ B cell levels totreatment with S239D/I332E rituximab. Data are presented as the averageof 3 monkeys/sample.

FIGS. 34 a and 34 c. AlphaScreen assay measuring binding of selectalemtuzumab (FIG. 34 a) and trastuzumab (FIG. 34 b) Fc variants to mouseFcγRIII, as described in Example 10. The binding data were normalized tothe upper and lower baselines for each particular antibody, and thecurves represent the fits of the data to a one site competition model.PBS was used as a negative control.

FIG. 35. Cell-based ADCC assays of select Fc variants in the context oftrastuzumab using mouse PBMCs as effector cells. ADCC was measured usingthe DELFIA® EuTDA-based cytotoxicity assay using Sk-Br-3 breastcarcinoma target cells and 8-fold excess mouse PBMCs. The bar graphshows the raw fluorescence data for the indicated trastuzumab antibodiesat 10 ng/ml. The PBMC bar indicates basal levels of cytotoxicity in theabsence of antibody, and TX indicates complete cell lysis in thepresence of Triton X1000.

FIG. 36. AlphaScreen assay measuring binding to human V158 FcγRIIIa byselect trastuzumab Fc variants expressed in 293T and CHO cells, asdescribed in Example 11. The binding data were normalized to the upperand lower baselines for each particular antibody, and the curvesrepresent the fits of the data to a one site competition model. PBS wasused as a negative control.

FIGS. 37 a-37 b. Synergy of Fc variants and engineered glycoforms. FIG.37 a presents an AlphaScreen assay showing V158 FcγRIIIa binding by WTand Fc variant (V209, S239/1332E/A330L) trastuzumab expressed in 293T,CHO, and Lec-13 CHO cells. The data were normalized to the upper andlower baselines for each antibody, and the curves represent the fits ofthe data to a one site competition model. PBS was used as a negativecontrol. FIG. 37 b presents a cell-based ADCC assay showing the abilityof 239T, CHO, and Lec-13 CHO expressed WT and V209 trastuzumab tomediate ADCC. ADCC was measured using the DELFIA® EuTDA-basedcytotoxicity assay as described previously, with Sk-Br-3 breastcarcinoma target cells. The data show the dose-dependence of ADCC onantibody concentration for the indicated trastuzumab antibodies,normalized to the minimum and maximum fluorescence signal for eachparticular curve, provided by the baselines at low and high antibodyconcentrations respectively. The curves represent the fits of the datato a sigmoidal dose-response model using nonlinear regression.

FIG. 38. AlphaScreen assay showing binding of aglycosylated alemtuzumabFc variants to human V158 FcγRIIIa. The binding data were normalized tothe upper and lower baselines for each particular antibody, and thecurves represent the fits of the data to a one site competition model.PBS was used as a negative control.

FIG. 39. AlphaScreen assay comparing human V158 FcγRIIIa binding byselect alemtuzumab Fc variants in glycosylated (solid symbols, solidlines) and deglycosylated (open symbols, dotted lines). The binding datawere normalized to the upper and lower baselines for each particularantibody, and the curves represent the fits of the data to a one sitecompetition model.

FIGS. 40 a-40 c. Sequences showing improved anti-CD20 antibodies. Thelight and heavy chain sequences of rituximab are presented in FIG. 40 a(SEQ ID NO: 5) and FIG. 40 b (SEQ ID NO: 6) respectively, and are takenfrom translated Sequence 3 of U.S. Pat. No. 5,736,137. Relevantpositions in FIG. 40 b are bolded, including S239, V240, V2641, H268,E272, K274, N297, S298, K326, A330, and 1332. FIG. 40 c (SEQ ID NO: 7)shows the improved anti-CD20 antibody heavy chain sequences, withvariable positions designated in bold as X1, X2, X3, X4, X5, X6, X7, X8,X9, Z1, and Z2. The table below the sequence provides possiblesubstitutions for these positions. The improved anti-CD20 antibodysequences comprise at least one non-WT amino acid selected from thegroup of possible substitutions for X1, X2, X3, X4, X5, X6, X7, X8, andX9. These improved anti-CD20 antibody sequences may also comprise asubstitution Z1 and/or Z2. These positions are numbered according to theEU index as in Kabat, and thus do not correspond to the sequential orderin the sequence.

FIG. 41 a-41 pp depicts the set of Fc variants that were constructed andexperimentally tested.

FIG. 42 depicts SEQ ID NO:8; the particular Xaa residues are as shown inTable 10.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “amino acid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. The preferredamino acid modification herein is a substitution. By “amino acidsubstitution” or “substitution” herein is meant the replacement of anamino acid at a particular position in a parent polypeptide sequencewith another amino acid. For example, the substitution 1332E refers to avariant polypeptide, in this case an Fc variant, in which the isoleucineat position 332 is replaced with a glutamic acid. In some embodiments,the WT identity need not be defined. For example, the substitution 332Erefers to a variant polypeptide in which position 332 is mutated toglutamic acid.

By “antibody” herein is meant a protein consisting of one or morepolypeptides substantially encoded by all or part of the recognizedimmunoglobulin genes. The recognized immunoglobulin genes, for examplein humans, include the kappa (κ), lambda (λ), and heavy chain geneticloci, which together comprise the myriad variable region genes, and theconstant region genes mu (μ), delta (δ), gamma (γ), sigma (σ), and alpha(α) which encode the IgM, IgD, IgG, IgE, and IgA isotypes respectively.Antibody herein is meant to include full length antibodies and antibodyfragments, and may refer to a natural antibody from any organism, anengineered antibody, or an antibody generated recombinantly forexperimental, therapeutic, or other purposes as further defined below.The term “antibody” includes antibody fragments, as are known in theart, such as Fab, Fab′, F(ab′)₂, Fv, scFv, or other antigen-bindingsubsequences of antibodies, either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAtechnologies. Particularly preferred are full length antibodies thatcomprise Fc variants as described herein. The term “antibody” comprisesmonoclonal and polyclonal antibodies. Antibodies can be antagonists,agonists, neutralizing, inhibitory, or stimulatory. The antibodies ofthe present invention may be nonhuman, chimeric, humanized, or fullyhuman, as described below in more detail.

Specifically included within the definition of “antibody” areaglycosylated antibodies. By “aglvcosvlated antibody” as used herein ismeant an antibody that lacks carbohydrate attached at position 297 ofthe Fc region, wherein numbering is according to the EU system as inKabat. The aglycosylated antibody may be a deglycosylated antibody, thatis an antibody for which the Fc carbohydrate has been removed, forexample chemically or enzymatically. Alternatively, the aglycosylatedantibody may be a nonglycosylated or unglycosylated antibody, that is anantibody that was expressed without Fc carbohydrate, for example bymutation of one or residues that encode the glycosylation pattern or byexpression in an organism that does not attach carbohydrates toproteins, for example bacteria.

Specifically included within the definition of “antibody” arefull-length antibodies that contain an Fc variant portion. By “fulllength antibody” herein is meant the structure that constitutes thenatural biological form of an antibody, including variable and constantregions. For example, in most mammals, including humans and mice, thefull length antibody of the IgG class is a tetramer and consists of twoidentical pairs of two immunoglobulin chains, each pair having one lightand one heavy chain, each light chain comprising immunoglobulin domainsV_(L) and C_(L), and each heavy chain comprising immunoglobulin domainsV_(H), Cγ1 (C_(H)1), Cγ2 (C_(H)2), and Cγ3 (C_(H)3). In some mammals,for example in camels and llamas, IgG antibodies may consist of only twoheavy chains, each heavy chain comprising a variable domain attached tothe Fc region. By “IqG” as used herein is meant a polypeptide belongingto the class of antibodies that are substantially encoded by arecognized immunoglobulin gamma gene. In humans this class comprisesIgG1, IgG2, IgG3, and IgG4. In mice this class comprises IgG1, IgG2a,IgG2b, IgG3.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids or any non-natural analogues thatmay be present at a specific, defined position. By “protein” herein ismeant at least two covalently attached amino acids, which includesproteins, polypeptides, oligopeptides and peptides. The protein may bemade up of naturally occurring amino acids and peptide bonds, orsynthetic peptidomimetic structures, i.e. “analogs”, such as peptoids(see Simon et al., 1992, Proc Natl Acad Sci USA 89(20):9367,incorporated by reference) particularly when LC peptides are to beadministered to a patient. Thus “amino acid”, or “peptide residue”, asused herein means both naturally occurring and synthetic amino acids.For example, homophenylalanine, citrulline and norleucine are consideredamino acids for the purposes of the invention. “Amino acid” alsoincludes imino acid residues such as proline and hydroxyproline. Theside chain may be in either the (R) or the (S) configuration. In thepreferred embodiment, the amino acids are in the (S) or L-configuration.If non-naturally occurring side chains are used, non-amino acidsubstituents may be used, for example to prevent or retard in vivodegradation.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC. By “effector cell” as used herein is meant a cellof the immune system that expresses one or more Fc receptors andmediates one or more effector functions. Effector cells include but arenot limited to monocytes, macrophages, neutrophils, dendritic cells,eosinophils, mast cells, platelets, B cells, large granular lymphocytes,Langerhans' cells, natural killer (NK) cells, and yy T cells, and may befrom any organism including but not limited to humans, mice, rats,rabbits, and monkeys. By “library” herein is meant a set of Fc variantsin any form, including but not limited to a list of nucleic acid oramino acid sequences, a list of nucleic acid or amino acid substitutionsat variable positions, a physical library comprising nucleic acids thatencode the library sequences, or a physical library comprising the Fcvariant proteins, either in purified or unpurified form.

By “Fc” or “Fc region”, as used herein is meant the polypeptidecomprising the constant region of an antibody excluding the firstconstant region immunoglobulin domain. Thus Fc refers to the last twoconstant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains. For IgA and IgM, Fc mayinclude the J chain. For IgG, as illustrated in FIG. 1, Fc comprisesimmunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hingebetween Cgammal (Cγ1) and Cgamma2 (Cγ2). Although the boundaries of theFc region may vary, the human IgG heavy chain Fc region is usuallydefined to comprise residues C226 or P230 to its carboxyl-terminus,wherein the numbering is according to the EU index as in Kabat. Fc mayrefer to this region in isolation, or this region in the context of anFc polypeptide, as described below. By “Fc polypeptide” as used hereinis meant a polypeptide that comprises all or part of an Fc region. Fcpolypeptides include antibodies, Fc fusions, isolated Fcs, and Fcfragments.

By “Fc fusion” as used herein is meant a protein wherein one or morepolypeptides or small molecules is operably linked to an Fc region or aderivative thereof. Fc fusion is herein meant to be synonymous with theterms “immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptorglobulin” (sometimes with dashes) as used in the prior art (Chamow etal., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr OpinImmunol 9:195-200. incorporated by reference). An Fc fusion combines theFc region of an immunoglobulin with a fusion partner, which in generalcan be any protein or small molecule. The role of the non-Fc part of anFc fusion, i.e. the fusion partner, may be to mediate target binding,and thus it is functionally analogous to the variable regions of anantibody.

By “Fc gamma receptor” or “FcγR” as used herein is meant any member ofthe family of proteins that bind the IgG antibody Fc region and aresubstantially encoded by the FcγR genes. In humans this family includesbut is not limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb,and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (includingallotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2),and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1and FcγRIIIb-NA2), as well as any undiscovered human FcγRs or FcγRisoforms or allotypes. An FcγR may be from any organism, including butnot limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

By “Fc ligand” or “effector ligand” as used herein is meant a molecule,preferably a polypeptide, from any organism that binds to the Fc regionof an antibody to form an Fc/Fc ligand complex. Binding of an Fc ligandto Fc preferably elicits or more effector functions. Fc ligands includebut are not limited to Fc receptors, FcγRs, FcαRs, FcεRs, FcRn, C1q, C3,mannan binding lectin, mannose receptor, staphylococcal protein A,streptococcal protein G, and viral FcγR. Fc ligands also include Fcreceptor homologs (FcRH), which are a family of Fc receptors that arehomologous to the FcγRs (Davis et al., 2002, Immunological Reviews190:123-136, incorporated by reference). Fc ligands may includeundiscovered molecules that bind Fc.

By “IqG” as used herein is meant a polypeptide belonging to the class ofantibodies that are substantially encoded by a recognized immunoglobulingamma gene. In humans this class comprises IgG1, IgG2, IgG3, and IgG4.In mice this class comprises IgG1, IgG2a, IgG2b, IgG3. By“immunoglobulin (Ig)” herein is meant a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes.Immunoglobulins include but are not limited to antibodies.Immunoglobulins may have a number of structural forms, including but notlimited to full length antibodies, antibody fragments, and individualimmunoglobulin domains. By “immunoqlobulin (Ig) domain” herein is meanta region of an immunoglobulin that exists as a distinct structuralentity as ascertained by one skilled in the art of protein structure. Igdomains typically have a characteristic β-sandwich folding topology. Theknown Ig domains in the IgG class of antibodies are V_(H), Cγ1, Cγ2,Cγ3, V_(L), and C_(L).

By “parent polypeptide” or “precursor polypeptide” (including Fc parentor precursors) as used herein is meant a polypeptide that issubsequently modified to generate a variant. Said parent polypeptide maybe a naturally occurring polypeptide, or a variant or engineered versionof a naturally occurring polypeptide. Parent polypeptide may refer tothe polypeptide itself, compositions that comprise the parentpolypeptide, or the amino acid sequence that encodes it. Accordingly, by“parent Fc polypeptide” as used herein is meant a Fc polypeptide that ismodified to generate a variant, and by “parent antibody” as used hereinis meant an antibody that is modified to generate a variant antibody.

As outlined above, certain positions of the Fc molecule can be altered.By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index as in Kabat. For example,position 297 is a position in the human antibody IgG1. Correspondingpositions are determined as outlined above, generally through alignmentwith other parent sequences.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297, also referred to as N297) is a residue in thehuman antibody IgG1.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. A targetantigen may be a protein, carbohydrate, lipid, or other chemicalcompound.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the V_(κ), V_(λ), and/or V_(H) genes that make up thekappa, lambda, and heavy chain immunoglobulin genetic loci respectively.

By “variant polypeptide” as used herein is meant a polypeptide sequencethat differs from that of a parent polypeptide sequence by virtue of atleast one amino acid modification. The parent polypeptide may be anaturally occurring or wild-type (WT) polypeptide, or may be a modifiedversion of a WT polypeptide. Variant polypeptide may refer to thepolypeptide itself, a composition comprising the polypeptide, or theamino sequence that encodes it. Preferably, the variant polypeptide hasat least one amino acid modification compared to the parent polypeptide,e.g. from about one to about ten amino acid modifications, andpreferably from about one to about five amino acid modificationscompared to the parent. The variant polypeptide sequence herein willpreferably possess at least about 80% homology with a parent polypeptidesequence, and most preferably at least about 90% homology, morepreferably at least about 95% homology. Accordingly, by “Fc variant” asused herein is meant an Fc sequence that differs from that of a parentFc sequence by virtue of at least one amino acid modification. An Fcvariant may only encompass an Fc region, or may exist in the context ofan antibody, Fc fusion, isolated Fc, Fc fragment, or other polypeptidethat is substantially encoded by Fc. Fc variant may refer to the Fcpolypeptide itself, compositions comprising the Fc variant polypeptide,or the amino acid sequence that encodes it.

The Fc variants of the present invention are defined according to theamino acid modifications that compose them. Thus, for example, I332E isan Fc variant with the substitution I332E relative to the parent Fcpolypeptide. Likewise, S239D/A330L/I332E (also referred to as239D/330L/332E) defines an Fc variant with the substitutions S239D,A330L, and I332E (239D, 330L, and 332E) relative to the parent Fcpolypeptide. It is noted that the order in which substitutions areprovided is arbitrary, that is to say that, for example,S239D/A330L/I332E is the same Fc variant as S239D/I332E/A330L, and soon. For all positions discussed in the present invention, numbering isaccording to the EU index or EU numbering scheme (Kabat et al., 1991,Sequences of Proteins of Immunological Interest, 5th Ed., United StatesPublic Health Service, National Institutes of Health, Bethesda,incorporated by reference). The EU index or EU index as in Kabat refersto the numbering of the EU antibody (Edelman et al., 1969, Proc NatlAcad Sci USA 63:78-85, incorporated by reference).

The present invention is directed to optimized Fc variants useful in avariety of contexts. As outlined above, current antibody therapiessuffer from a variety of problems. The present invention provides apromising means for enhancing the anti-tumor potency of antibodies isvia enhancement of their ability to mediate cytotoxic effector functionssuch as ADCC, ADCP, and CDC. The present invention shows that antibodieswith an Fc region optimized for binding to certain FcγRs may bettermediate effector functions and thereby destroy cancer cells moreeffectively in patients. The balance between activating and inhibitingreceptors is an important consideration, and optimal effector functionmay result from an Fc with enhanced affinity for activation receptors,for example FcγRI, FcγRIIa/c, and FcγRIIIa, yet reduced affinity for theinhibitory receptor FcγRIIb. Furthermore, because FcγRs can mediateantigen uptake and processing by antigen presenting cells, enhancedFc/FcγR affinity may also improve the capacity of antibody therapeuticsto elicit an adaptive immune response. For example, several mutationsdisclosed in this application, including S298A, E333A, and K334A, showenhanced binding to the activating receptor FcγRIIIa and reduced bindingto the inhibitory receptor FcγRIIb. These mutations maybe combined toobtain double and triple mutation variants that show additiveimprovements in binding. A particular variant is a S298A/E333A/K334Atriple mutant with approximately a 1.7-fold increase in binding to F158FcγRIIIa, a 5-fold decrease in binding to FcγRIIb, and a 2.1-foldenhancement in ADCC.

Although there is a need for greater effector function, for someantibody therapeutics reduced or eliminated effector function may bedesired. This is often the case for therapeutic antibodies whosemechanism of action involves blocking or antagonism but not killing ofthe cells bearing target antigen. In these cases depletion of targetcells is undesirable and can be considered a side effect. For example,the ability of anti-CD4 antibodies to block CD4 receptors on T cellsmakes them effective anti-inflammatories, yet their ability to recruitFcγR receptors also directs immune attack against the target cells,resulting in T cell depletion (Reddy et al., 2000, J Immunol164:1925-1933, incorporated by reference). Effector function can also bea problem for radiolabeled antibodies, referred to as radioconjugates,and antibodies conjugated to toxins, referred to as immunotoxins. Thesedrugs can be used to destroy cancer cells, but the recruitment of immunecells via Fc interaction with FcγRs brings healthy immune cells inproximity to the deadly payload (radiation or toxin), resulting indepletion of normal lymphoid tissue along with targeted cancer cells(Hutchins et al., 1995, Proc Natl Acad Sci USA 92:11980-11984; White etal., 2001, Annu Rev Med 52:125-145, incorporated by reference). Thisproblem can potentially be circumvented by using IgG isotypes thatpoorly recruit complement or effector cells, for example IgG2 and IgG4.An alternate solution is to develop Fc variants that reduce or ablatebinding (Alegre et al., 1994, Transplantation 57:1537-1543; Hutchins etal., 1995, Proc Natl Acad Sci USA 92:11980-11984; Armour et al., 1999,Eur J Immunol 29:2613-2624; Reddy et al., 2000, J Immunol 164:1925-1933;Xu et al., 2000, Cell Immunol 200:16-26; Shields et al., 2001, J BiolChem 276:6591-6604) (U.S. Pat. No. 6,194,551; U.S. Pat. No. 5,885,573;PCT WO 99/58572), all incorporated by reference. A criticalconsideration for the reduction or elimination of effector function isthat other important antibody properties not be perturbed. Fc variantsshould be engineered that not only ablate binding to FcγRs and/or C1q,but also maintain antibody stability, solubility, and structuralintegrity, as well as ability to interact with other important Fcligands such as FcRn and proteins A and G.

In addition, the invention utilizes engineered glycoforms that canenhance Fc/FcγR affinity and effector function. An aglycosylated Fc withfavorable solution properties and the capacity to mediate effectorfunctions would be significantly enabling for the alternate productionmethods described above. By overcoming the structural and functionalshortcomings of aglycosylated Fc, antibodies can be produced in bacteriaand transgenic plants and animals with reduced risk of immunogenicity,and with effector function for clinical applications in whichcytotoxicity is desired such as cancer. The present invention describesthe utilization of protein engineering methods to develop stable,soluble Fc variants with effector function. Currently, such Fc variantsdo not exist in the art.

Fc Variants of the Present Invention

The Fc variants of the present invention may find use in a variety of Fcpolypeptides. An Fc polypeptide that comprises an Fc variant of thepresent invention is herein referred to as an “Fc polypeptide of thepresent invention”. Fc polypeptides of the present invention includepolypeptides that comprise the Fc variants of the present invention inthe context of a larger polypeptide, such as an antibody or Fc fusion.That is, Fc polypeptides of the present invention include antibodies andFc fusions that comprise Fc variants of the present invention. By“antibody of the present invention” as used herein is meant an antibodythat comprises an Fc variant of the present invention. By “Fc fusion ofthe present invention” as used herein refers to an Fc fusion thatcomprises an Fc variant of the present invention. Fc polypeptides of thepresent invention also include polypeptides that comprise little or noadditional polypeptide sequence other than the Fc region, referred to asan isolated Fc. By “isolated Fc of the present invention” used herein ismeant an Fc polypeptide that comprises an Fc variant of the presentinvention, and comprises little or no additional polypeptide sequenceother than the Fc region. Fc polypeptides of the present invention alsoinclude fragments of the Fc region. By “Fc fragment of the presentinvention” as used herein is meant an Fc fragment that comprises an Fcvariant of the present invention. As described below, any of theaforementioned Fc polypeptides of the present invention may be fused toone or more fusion partners or conjugate partners to provide desiredfunctional properties.

Fc variants may be constructed in a parent Fc polypeptide irrespectiveof its context. That is to say that, the sole criteria for a parent Fcpolypeptide is that it comprise an Fc region. The parent Fc polypeptidesdescribed herein may be derived from a wide range of sources, and may besubstantially encoded by one or more Fc genes from any organism,including but not limited to humans, rodents including but not limitedto mice and rats, lagomorpha such as rabbits and hares, camelidae suchas camels, llamas, and dromedaries, and non-human primates, includingbut not limited to Prosimians, Platyrrhini (New World monkeys),Cercopithecoidea (Old World monkeys), and Hominoidea include theGibbons, Lesser and Great Apes, with humans most preferred. The parentFc polypeptides of the present invention may be substantially encoded byimmunoglobulin genes belonging to any of the antibody classes, includingbut not limited to sequences belonging to the IgG (including humansubclasses IgG1, IgG2, IgG3, or IgG4), IgA (including human subclassesIgA1 and IgA2), IgD, IgE, IgG, or IgM classes of antibodies. Mostpreferably the parent Fc polypeptides of the present invention comprisesequences belonging to the human IgG class of antibodies. For example,the parent Fc polypeptide may be a parent antibody, for example a humanIgG1 antibody, a human IgA antibody, or a mouse IgG2a or IgG2b antibody.Said parent antibody may be nonhuman, chimeric, humanized, or fullyhuman as described in detail below. The parent Fc polypeptide may bemodified or engineered in some way, for example a parent antibody may beaffinity matured, or may possess engineered glycoforms, all as describedmore fully below. Alternatively, the parent Fc polypeptide may be an Fcfusion, for example an Fc fusion wherein the fusion partner targets acell surface receptor. Alternatively, the parent Fc polypeptide may bean isolated Fc region, comprising little or no other polypeptidesequence outside the Fc region. The parent Fc polypeptide may be anaturally existing Fc region, or may be an existing engineered variantof an Fc polypeptide. What is important is that the parent Fcpolypeptide comprise an Fc region, which can then be mutated to generatean Fc variant.

The Fc variants of the present invention may be an antibody, referred toherein as an “antibody of the present invention”. Antibodies of thepresent invention may comprise immunoglobulin sequences that aresubstantially encoded by immunoglobulin genes belonging to any of theantibody classes, including but not limited to IgG (including humansubclasses IgG1, IgG2, IgG3, or IgG4), IgA (including human subclassesIgA1 and IgA2), IgD, IgE, IgG, and IgM classes of antibodies. Mostpreferably the antibodies of the present invention comprise sequencesbelonging to the human IgG class of antibodies. Antibodies of thepresent invention may be nonhuman, chimeric, humanized, or fully human.As will be appreciated by one skilled in the art, these different typesof antibodies reflect the degree of “humanness” or potential level ofimmunogenicity in a human. For a description of these concepts, seeClark et al., 2000 and references cited therein (Clark, 2000, ImmunolToday 21:397-402, incorporated by reference). Chimeric antibodiescomprise the variable region of a nonhuman antibody, for example V_(H)and V_(L) domains of mouse or rat origin, operably linked to theconstant region of a human antibody (see for example U.S. Pat. No.4,816,567, incorporated by reference). Said nonhuman variable region maybe derived from any organism as described above, preferably mammals andmost preferably rodents or primates. In one embodiment, the antibody ofthe present invention comprises monkey variable domains, for example asdescribed in Newman et al., 1992, Biotechnology 10:1455-1460, U.S. Pat.No. 5,658,570, and U.S. Pat. No. 5,750,105, incorporated by reference.In a preferred embodiment, the variable region is derived from anonhuman source, but its immunogenicity has been reduced using proteinengineering. In a preferred embodiment, the antibodies of the presentinvention are humanized (Tsurushita & Vasquez, 2004, Humanization ofMonoclonal Antibodies, Molecular Biology of B Cells, 533-545, ElsevierScience (USA), incorporated by reference). By “humanized” antibody asused herein is meant an antibody comprising a human framework region(FR) and one or more complementarity determining regions (CDR's) from anon-human (usually mouse or rat) antibody. The non-human antibodyproviding the CDR's is called the “donor” and the human immunoglobulinproviding the framework is called the “acceptor”. Humanization reliesprincipally on the grafting of donor CDRs onto acceptor (human) V_(L)and V_(H) frameworks (Winter U.S. Pat. No. 5,225,539, incorporated byreference). This strategy is referred to as “CDR grafting”.“Backmutation” of selected acceptor framework residues to thecorresponding donor residues is often required to regain affinity thatis lost in the initial grafted construct (U.S. Pat. No. 5,530,101; U.S.Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762;U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat. No.5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213,incorporated by reference). A large number of other methods forhumanization are known in the art (Tsurushita & Vasquez, 2004,Humanization of Monoclonal Antibodies, Molecular Biology of B Cells,533-545, Elsevier Science (USA), incorporated by reference), and any ofsuch methods may find use in the present invention for modifying Fcvariants for reduced immunogenicity. The humanized antibody optimallyalso will comprise at least a portion of an immunoglobulin constantregion, typically that of a human immunoglobulin, and thus willtypically comprise a human Fc region. In a most preferred embodiment,the immunogenicity of an Fc variant of the present invention is reducedusing a method described in U.S. Ser. No. 11/004,590, filed Dec. 3,2004, entitled “Methods of Generating Variant Proteins with IncreasedHost String Content and Compositions Thereof,” incorporated byreference. In an alternate embodiment, the antibodies of the presentinvention may be fully human, that is the sequences of the antibodiesare completely or substantially human. A number of methods are known inthe art for generating fully human antibodies, including the use oftransgenic mice (Bruggemann et al., 1997, Curr Opin Biotechnol8:455-458, incorporated by reference) or human antibody librariescoupled with selection methods (Griffiths et al., 1998, Curr OpinBiotechnol 9:102-108, incorporated by reference).

The Fc variants of the present invention may be an Fc fusion, referredto herein as an “Fc fusion of the present invention”. Fc fusions of thepresent invention comprise an Fc polypeptide operably linked to one ormore fusion partners. The role of the fusion partner typically, but notalways, is to mediate binding of the Fc fusion to a target antigen.(Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al.,1997, Curr Opin Immuno/9:195-200, incorporated by reference). Forexample, the approved drug alefacept (marketed as AMEVIVE®) is animmunosuppressive Fc fusion that consists of the extracellularCD2-binding portion of the human leukocyte function antigen-3 (LFA-3)linked to the Fc region of human IgG1. The approved drug etanercept(marketed as ENBREL®) is an Fc fusion comprising the extracellularligand-binding portion of human tumor necrosis factor receptor (TNFR)linked to human IgG1 Fc. Virtually any protein, polypeptide, peptide, orsmall molecule may be linked to Fc to generate an Fc fusion. Fusionpartners include but are not limited to receptors and extracellularreceptor domains, adhesion molecules, ligands, enzymes, cytokines,chemokines, or some other protein or protein domain. The fusion partnermay also play a role as a chemoattractant. Undiscovered ligands orreceptors may serve as fusion partners for the Fc variants of thepresent invention. Small molecules may serve as fusion partners, and mayinclude any therapeutic agent that directs the Fc fusion to atherapeutic target. Such targets may be any molecule, preferrably anextracellular receptor, that is implicated in disease. Two families ofsurface receptors that are targets of a number of approved smallmolecule drugs are G-Protein Coupled Receptors (GPCRs), and ionchannels, including K+, Na+, Ca+ channels. Nearly 70% of all drugscurrently marketed worldwide target GPCRs. Thus the Fc variants of thepresent invention may be fused to a small molecule that targets, forexample, one or more GABA receptors, purinergic receptors, adrenergicreceptors, histaminergic receptors, opiod receptors, chemokinereceptors, glutamate receptors, nicotinic receptors, the 5HT (serotonin)receptor, and estrogen receptors. A fusion partner may be asmall-molecule mimetic of a protein that targets a therapeuticallyuseful target. Specific examples of particular drugs that may serve asFc fusion partners can be found in L. S. Goodman et al., Eds., Goodmanand Gilman's The Pharmacological Basis of Therapeutics (McGraw-Hill, NewYork, ed. 9, 1996, incorporated by reference). Fusion partners includenot only small molecules and proteins that bind known targets forexisting drugs, but orphan receptors that do not yet exist as drugtargets. The completion of the genome and proteome projects are provingto be a driving force in drug discovery, and these projects have yieldeda trove of orphan receptors. There is enormous potential to validatethese new molecules as drug targets, and develop protein and smallmolecule therapeutics that target them. Such protein and small moleculetherapeutics are contemplated as Fc fusion partners that employ the Fcvariants of the present invention. Fc fusions of the invention maycomprise immunoglobulin sequences that are substantially encoded byimmunoglobulin genes belonging to any of the antibody classes, includingbut not limited to IgG (including human subclasses IgG1, IgG2, IgG3, orIgG4), IgA (including human subclasses IgA1 and IgA2), IgD, IgE, IgG,and IgM classes of antibodies. Most preferably the Fc fusions of thepresent invention comprise sequences belonging to the human IgG class ofantibodies. A variety of linkers, defined and described below, may beused to covalently link Fc to a fusion partner to generate an Fc fusion.

The Fc variants of the present invention may find use in an isolated Fc,that is an Fc polypeptide that comprises little or no additionalpolypeptide sequence other than the Fc region and that comprises an Fcvariant of the present invention. Isolated Fc of the present inventionare meant as molecules wherein the desired function of the molecule, forexample the desired therapeutic function, resides solely in the Fcregion. Thus the therapeutic target of an isolated Fc of the presentinvention is likely to involve one or more Fc ligands. An isolated Fcthat comprises the Fc variant may require no additional covalentpolypeptide sequence to achieve its desired outcome. In a preferredembodiment, said isolated Fc comprises from 90-100% of the Fc region,with little or no “extra” sequence. Thus, for example, an isolated Fc ofthe present invention may comprise residues C226 or P230 to thecarboxyl-terminus of human IgG1, wherein the numbering is according tothe EU index as in Kabat. In one embodiment, the isolated Fc of thepresent invention may contain no extra sequence outside the Fc region.However it is also contemplated that isolated Fc's may not also compriseadditional polypeptide sequences. For example, an isolated Fc may, inaddition to comprising an Fc variant Fc region, comprise additionalpolypeptide sequence tags that enable expression, purification, and thelike.

The Fc variants of the present invention may find use in a fragment ofthe Fc region, that is an Fc polypeptide that comprises an Fc fragmentthat comprises an Fc variant of the present invention. Clearly arequirement of an Fc fragment of the present invention is that itcontains the position(s) at which the amino acid modifications of the Fcvariant are made. An Fc fragment of the present invention may comprisefrom 1-90% of the Fc region, with 10-90% being preferred, and 30-90%being most preferred. Thus for example, an Fc fragment of the presentinvention may comprise an Fc variant IgG1 Cγ2 domain, an Fc variant IgG1Cγ2 domain and hinge region, an Fc variant IgG1 Cγ3 domain, and soforth. In one embodiment, an Fc fragment of the present inventionadditionally comprises a fusion partner, effectively making it an Fcfragment fusion. As with isolated Fcs, Fc fragments may or may notcontain extra polypeptide sequence.

Fc variants of the present invention may be substantially encoded bygenes from any organism, preferably mammals, including but not limitedto humans, rodents including but not limited to mice and rats,lagomorpha including but not limited to rabbits and hares, camelidaeincluding but not limited to camels, llamas, and dromedaries, andnon-human primates, including but not limited to Prosimians, Platyrrhini(New World monkeys), Cercopithecoidea (Old World monkeys), andHominoidea including the Gibbons and Lesser and Great Apes. In a mostpreferred embodiment, the Fc variants of the present invention aresubstantially human. The Fc variants of the present invention may besubstantially encoded by immunoglobulin genes belonging to any of theantibody classes. In a most preferred embodiment, the Fc variants of thepresent invention comprise sequences belonging to the IgG class ofantibodies, including human subclasses IgG1, IgG2, IgG3, and IgG4. In analternate embodiment, the Fc variants of the present invention comprisesequences belonging to the IgA (including human subclasses IgA1 andIgA2), IgD, IgE, IgG, or IgM classes of antibodies. The Fc variants ofthe present invention may comprise more than one protein chain. That is,the present invention may find use in an Fc variant that is a monomer oran oligomer, including a homo- or hetero-oligomer.

In the most preferred embodiment, the Fc polypeptides of the inventionare based on human IgG sequences, and thus human IgG sequences are usedas the “base” sequences against which other sequences are compared,including but not limited to sequences from other organisms, for examplerodent and primate sequences, as well as sequences from otherimmunoglobulin classes such as IgA, IgE, IgGD, IgGM, and the like. It iscontemplated that, although the Fc variants of the present invention areengineered in the context of one parent Fc variant, the variants may beengineered in or “transferred” to the context of another, second parentFc variant. This is done by determining the “equivalent” or“corresponding” residues and substitutions between the first and secondFc variants, typically based on sequence or structural homology betweenthe sequences of the two Fc variants. In order to establish homology,the amino acid sequence of a first Fc variant outlined herein isdirectly compared to the sequence of a second Fc variant. After aligningthe sequences, using one or more of the homology alignment programsknown in the art (for example using conserved residues as betweenspecies), allowing for necessary insertions and deletions in order tomaintain alignment (i.e., avoiding the elimination of conserved residuesthrough arbitrary deletion and insertion), the residues equivalent toparticular amino acids in the primary sequence of the first Fc variantare defined. Alignment of conserved residues preferably should conserve100% of such residues. However, alignment of greater than 75% or aslittle as 50% of conserved residues is also adequate to defineequivalent residues. Equivalent residues may also be defined bydetermining structural homology between a first and second Fc variantthat is at the level of tertiary structure for Fc variants whosestructures have been determined. In this case, equivalent residues aredefined as those for which the atomic coordinates of two or more of themain chain atoms of a particular amino acid residue of the parent orprecursor (N on N, CA on CA, C on C and O on O) are within 0.13 nm andpreferably 0.1 nm after alignment. Alignment is achieved after the bestmodel has been oriented and positioned to give the maximum overlap ofatomic coordinates of non-hydrogen protein atoms of the proteins.Regardless of how equivalent or corresponding residues are determined,and regardless of the identity of the parent Fc variant in which the Fcvariants are made, what is meant to be conveyed is that the Fc variantsdiscovered by the present invention may be engineered into any secondparent Fc variant that has significant sequence or structural homologywith said Fc variant. Thus for example, if a variant antibody isgenerated wherein the parent antibody is human IgG1, by using themethods described above or other methods for determining equivalentresidues, said variant antibody may be engineered in a human IgG2 parentantibody, a human IgA parent antibody, a mouse IgG2a or IgG2b parentantibody, and the like. Again, as described above, the context of theparent Fc variant does not affect the ability to transfer the Fcvariants of the present invention to other parent Fc variants. Forexample, the variant antibodies that are engineered in a human IgG1antibody that targets one epitope may be transferred into a human IgG2antibody that targets a different epitope, into an Fc fusion thatcomprises a human IgG1 Fc region that targets yet a different epitope,and so forth.

The Fc variants of the present invention may find use in a wide range ofproducts. In one embodiment the Fc variant of the invention is atherapeutic, a diagnostic, or a research reagent, preferably atherapeutic. Alternatively, the Fc variant of the present invention maybe used for agricultural or industrial uses. An antibody of the presentinvention may find use in an antibody composition that is monoclonal orpolyclonal. The Fc variants of the present invention may be agonists,antagonists, neutralizing, inhibitory, or stimulatory. In a preferredembodiment, the Fc variants of the present invention are used to killtarget cells that bear the target antigen, for example cancer cells. Inan alternate embodiment, the Fc variants of the present invention areused to block, antagonize, or agonize the target antigen. In analternately preferred embodiment, the Fc variants of the presentinvention are used to block, antagonize, or agonize the target antigenand kill the target cells that bear the target antigen.

Targets

Virtually any antigen may be targeted by the Fc variants of the presentinvention, including but not limited to proteins, subunits, domains,motifs, and/or epitopes belonging to the following list of targets:17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 AdenosineReceptor, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B,Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, ActivinRIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8,ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressins, aFGF, ALCAM, ALK, ALK-1,ALK-7, alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1,APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, Atrialnatriuretic factor, av/b3 integrin, Axl, b2M, B7-1, B7-2, B7-H,B-lymphocyte Stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1,BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM,BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b,BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF,BOK, Bombesin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC,complement factor 3 (C3), C3a, C4, C5, C5a, C10, CAl25, CAD-8,Calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associatedantigen, Cathepsin A, Cathepsin B, Cathepsin C/DPPI, Cathepsin D,Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S,Cathepsin V, Cathepsin X/Z/P, CBL, CCl, CCK2, CCL, CCL1, CCL11, CCL12,CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21,CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6,CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8,CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20,CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54,CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123,CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR,cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin,CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK,CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5,CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14,CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decayaccelerating factor, des(1-3)-IGF-1 (brain IGF-1), Dhh, digoxin, DNAM-1,Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR(ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, Enkephalinase, eNOS,Eot, eotaxin1, EpCAM, Ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1,Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activationprotein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3,FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Folliclestimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6,FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1,GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7(BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF,GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut4, glycoprotein IIb/IIIa (GP IIb/IIIa)m GM-CSF, gp130, gp72, GRO, Growthhormone releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gBglycoprotein, HSV gD glycoprotein, HGFA, High molecular weightmelanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin,human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309,IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF,IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R,IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha,INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain,Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrinalpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5(alphaV), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6,integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE,Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12,Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, KallikreinL3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5,LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bpi, LBP, LDGF,LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3,Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b,LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotoxin BetaReceptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF,MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC(HLA-DR), MIF, MIG,MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13,MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo,MSK, MSP, mucin (Mud), MUC18, Muellerian-inhibitin substance, Mug, MuSK,NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin,Neurotrophin-3, -4, or -6, Neurturin, Neuronal growth factor (NGF),NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN,OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP,PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4,PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP),P1GF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA,prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51,RANK, RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin,respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors,RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3,Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72),TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT,TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkalinephosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific,TGF-beta R1 (ALK-5), TGF-beta R11, TGF-beta RIIb, TGF-beta RIII,TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, ThymusCk-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor,TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc,TNF-R1, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAILR2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3DcR1, LIT, TRID),TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF R1CD120a, p55-60), TNFRSF1B(TNF R11CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFCR), TNFRSF4 (OX40 ACT35, TXGP1R), TNFRSF5 (CD40 p50), TNFRSF6 (FasApo-1, APT1, CD95), TNFRSF6B (DcR3M68, TR6), TNFRSF7 (CD27), TNFRSF8(CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAILR2TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD,TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand, TL2), TNFSF11(TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand, DR3Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK,TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18(GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-a Conectin, DIF, TNFSF2),TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 Ligandgp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3, TRAP),TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27 LigandCD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137 Ligand),TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associatedantigen CA 125, tumor-associated antigen expressing Lewis Y relatedcarbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1,VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, vonWillebrands factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B,WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,and receptors for hormones and growth factors.

One skilled in the art will appreciate that the aforementioned list oftargets refers not only to specific proteins and biomolecules, but thebiochemical pathway or pathways that comprise them. For example,reference to CTLA-4 as a target antigen implies that the ligands andreceptors that make up the T cell co-stimulatory pathway, includingCTLA-4, B7-1, B7-2, CD28, and any other undiscovered ligands orreceptors that bind these proteins, are also targets. Thus target asused herein refers not only to a specific biomolecule, but the set ofproteins that interact with said target and the members of thebiochemical pathway to which said target belongs. One skilled in the artwill further appreciate that any of the aforementioned target antigens,the ligands or receptors that bind them, or other members of theircorresponding biochemical pathway, may be operably linked to the Fcvariants of the present invention in order to generate an Fc fusion.Thus for example, an Fc fusion that targets EGFR could be constructed byoperably linking an Fc variant to EGF, TGF-β, or any other ligand,discovered or undiscovered, that binds EGFR. Accordingly, an Fc variantof the present invention could be operably linked to EGFR in order togenerate an Fc fusion that binds EGF, TGF-β, or any other ligand,discovered or undiscovered, that binds EGFR. Thus virtually anypolypeptide, whether a ligand, receptor, or some other protein orprotein domain, including but not limited to the aforementioned targetsand the proteins that compose their corresponding biochemical pathways,may be operably linked to the Fc variants of the present invention todevelop an Fc fusion.

Choosing the right target antigen for antibody therapy is a complexprocess and encompasses many variables. For anti-cancer treatment it isdesirable to have a target whose expression is restricted to thecancerous cells. Some targets that have proven especially amenable toantibody therapy are those with signaling functions. Other therapeuticantibodies exert their effects by blocking signaling of the receptor byinhibiting the binding between a receptor and it's cognate ligand.Another mechanism of action of therapeutic antibodies is to causereceptor down regulation. Although many therapeutically effectiveantibodies work in part by signaling through their target antigen, thisis not always the case. For example, some target classes such as cellsurface glycoforms do not generate any biological signal. However,altered glycoforms are often associated with disease states such ascancer. Another significant target type are those that internalizeeither as a normal function or in response to antibody binding. In thecase of targets that are soluble rather than cell surface bound therecruitment of effector functions would not result in any cell death.

Some targets that have proven especially amenable to antibody therapyare those with signalling functions. For example, antibody cross-linkingof the Her2/neu antigen may generate an apoptotic signal that results incancer cell death. In some cases such as the CD30 antigen, thisclustering with free antibody may be insufficient to cause apoptosis invitro. For in vitro assays sufficient clustering can be mediated bycrosslinking the antibody or by immobilizing it at high density to asurface such as the well of a microtiter plate. However, in vivo thiseffect may be mediated by binding of the antibody to the Fc ligands, forexample FcγRs expressed on a nearby cell. Antibody Fc variants that bindmore tightly to Fc ligands may thus more effectively cluster thesignaling target and lead to enhanced induction of apoptosis. Such amechanism could be tested experimentally by adding antibody with andwithout enhanced Fc ligand binding to cells expressing the desiredtarget that signals, and/or adding an Fc receptor and a correspondingantibody that will cluster the Fc receptor. Alternative means forclustering Fc receptor include immobilization on beads, andover-expression in a non-effector cell line. After allowing apoptosis tooccur, measurement of the relative apoptosis of target expressing cellswould enable a quantitiative determination of the effect.

Antibodies that cause cell death through their interaction with targetsmay have an additional benefit. The signals released by such dying cellsattract macrophages and other cells of the immune system. These cellscan then takeup the dead or dying cells in an antibody mediated manner.This has been shown to result in cross-presentation of antigen and thepotential for a host immune response against the target cells. Suchauto-antibodies in response to antibody therapy have been reported forthe antigen targets Her2 and CD20. For this reason it may beadvantageous to have Fc variants with altered receptor specificities tospecifically stimulate cross-presentation and an immune response ratherthan the undesired effect of tolerance induction.

Other therapeutic antibodies exert their effects by inhibitinginteraction between a receptor and it's cognate ligand, ultimatelyblocking signaling of the receptor. Such antibodies are used to treatmany disease states. In this case it may be advantageous to utilizeantibodies that do not recruit any host immune functions. A secondaryeffect of such an antibody may be actually inducing signalling itselfthrough receptor clustering. In this case the desired therapeutic effectof blocking signaling would be abrogated by antibody mediated signaling.As discussed above, this clustering may be enhanced by antibodyinteraction with cells containing an Fc receptor. In this case, use ofan Fc variant that binds less tightly or not at all to the Fc receptorwould be preferable. Such an antibody would not mediate signaling, andits mechanism of action would thereby be restricted to blockage ofreceptor/ligand interactions. Signaling receptors for which this wouldbe most appropriate would likely be monomeric receptors which can onlybe dimerized but not substantially clustered by a primary antibody.Mulitimeric receptors may be significantly clustered by the primaryantibody and may not require additional clustering by Fc receptorbinding.

Another potential mechanism of action of therapeutic antibodies isreceptor downregulation. Such may be the case, for example, with theinsulin-like growth factor receptor. Cell growth depends on continuedsignaling through the receptor, whereas in its absence cells cease togrow. One effect of antibodies directed against this receptor is todownregulate its expression and thereby ablate signaling. Cell recoveryfrom cytotoxic therapy requires stimulation of this receptor.Downregulation of this receptor prevents these cells from recovery andrenders the cytotoxic therapy substantially more effective. Forantibodies for which this is the primary mechanism of action, decreasedFc receptor binding may prevent the sequestration of antibody bynontarget binding to Fc receptors.

Although many therapeutically effective antibodies work in part bysignaling through their target antigen, this is not always the case. Forexample, some target classes such as cell surface glycoforms do notgenerate any biological signal. However, altered glycoforms are oftenassociated with disease states such as cancer. In other cases,interaction of antibodies with different epitopes of the same targetantigen may confer different signaling effects. In such cases where thisis little or no elicited signaling by binding of antibody or Fc fusionto target antigen, Fc polypeptides of the present invention may findutility in providing novel mechanisms of efficacy for otherwisenon-efficacious molecules.

One approach that has been taken in generating therapeutic antibodies tosuch nonsignaling targets is to couple the antibody to a cytotoxic agentsuch as a radio-isotope, toxin, or an enzyme that will process asubstrate to produce a cytotoxic agent in the vicinity of the tumor. Asan alternative to a cytotoxic moiety, Fc variants of the presentinvention may provide increased recruitment of immune functions that areinherently less toxic to the host while still effective at destroyingtarget cancer cells. Such Fc variants may, for example, be moreefficient at recruiting NK cells or at activating phagocytosis orinitiating CDC. Alternatively, if a cytotoxic agent is utilized, it maybe advantageous to use an Fc variant that provides reduced or altered Fcligand binding. This may reduce or ablate the cytotoxic effects of theagent on immune cells that express Fc receptors, thereby reducingtoxicity to the patient. Furthermore, reduction of Fc ligand binding mayhelp to minimize the generation of an immune response to the toxic agentor enzyme. As mentioned above, cell death may result in recruitment ofhost immune cells; antibody mediated cross-presentation in such a casemay be increased with immune response rather than immune tolerance if inaddition to a cytotoxic moiety the therapeutic antibody has increased Fcreceptor binding affinity or altered receptor specificity.

Another significant target type are those targets that internalize,either as a normal part of their biological function or in response toantibody binding. For such targets, many efforts have been made tocouple cytotoxic agents such as RNase, ricin and calicheamicin, whichcan only exert their effect after internalization. For such reagents, Fcligand binding may reduce efficacy due to nonproductive sequestration ofthe therapeutic by Fc ligands. In this case it may be advantageous toutilize Fc variants that provide decreased Fc ligand affinity.Conversely, antibody pre-association with Fc ligands prior to theirbinding to target antigen presented on cells may serve to inhibitinternalization of the target. In this case, increased Fc ligandaffinity may serve to improve pre-association and thereby recruitment ofeffector cells and the host immune response.

In the case of targets that are soluble rather than cell surface bound,recruitment of effector functions would not result directly in celldeath. However, there may be utility in stimulating the generation ofhost antibodies to the target. For some disease states, successfultreatment may require administration of the therapeutic antibody forextremely long periods of time. Such therapy may be prohibitively costlyor cumbersome. In these cases, stimulation of the host immune responseand the generation of antibodies may result in improved efficacy of thetherapeutic. This may be applicable as an adjuvant to vaccine therapy.Antibody Fc variants that mediate such an effect may have increasedaffinity for Fc ligands or altered Fc ligand specificity.

A number of antibodies and Fc fusions that are approved for use, inclinical trials, or in development may benefit from the Fc variants ofthe present invention. These antibodies and Fc fusions are hereinreferred to as “clinical products and candidates”. Thus in a preferredembodiment, the Fc polypeptides of the present invention may find use ina range of clinical products and candidates. For example, a number ofantibodies that target CD20 may benefit from the Fc polypeptides of thepresent invention. For example the Fc polypeptides of the presentinvention may find use in an antibody that is substantially similar torituximab (Rituxan®, IDEC/Genentech/Roche) (see for example U.S. Pat.No. 5,736,137), a chimeric anti-CD20 antibody approved to treatNon-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currently beingdeveloped by Genmab, an anti-CD20 antibody described in U.S. Pat. No.5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics,Inc.), HumaLYM (Intracel), and PRO70769 (PCT/US2003/040426, entitled“Immunoglobulin Variants and Uses Thereof”). A number of antibodies thattarget members of the family of epidermal growth factor receptors,including EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), Her4(ErbB-4), may benefit from the Fc polypeptides of the present invention.For example the Fc polypeptides of the present invention may find use inan antibody that is substantially similar to trastuzumab (Herceptin®,Genentech) (see for example U.S. Pat. No. 5,677,171), a humanizedanti-Her2/neu antibody approved to treat breast cancer; pertuzumab(rhuMab-2C4, Omnitarg™), currently being developed by Genentech; ananti-Her2 antibody described in U.S. Pat. No. 4,753,894; cetuximab(Erbitux®, Imclone) (U.S. Pat. No. 4,943,533; PCT WO 96/40210), achimeric anti-EGFR antibody in clinical trials for a variety of cancers;ABX-EGF (U.S. Pat. No. 6,235,883), currently being developed byAbgenix-Immunex-Amgen; HuMax-EGFr (U.S. Ser. No. 10/172,317), currentlybeing developed by Genmab; 425, EMD55900, EMD62000, and EMD72000 (MerckKGaA) (U.S. Pat. No. 5,558,864; Murthy et al. 1987, Arch BiochemBiophys. 252(2):549-60; Rodeck et al., 1987, J Cell Biochem.35(4):315-20; Kettleborough et al., 1991, Protein Eng. 4(7):773-83);ICR62 (Institute of Cancer Research) (PCT WO 95/20045; Modjtahedi etal., 1993, J. Cell Biophys. 1993, 22(1-3):129-46; Modjtahedi et al.,1993, Br J Cancer. 1993, 67(2):247-53; Modjtahedi et al, 1996, Br JCancer, 73(2):228-35; Modjtahedi et al, 2003, Int J Cancer,105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro deImmunologia Molecular, Cuba (U.S. Pat. No. 5,891,996; U.S. Pat. No.6,506,883; Mateo et al, 1997, Immunotechnology, 3(1):71-81); mAb-806(Ludwig Institute for Cancer Research, Memorial Sloan-Kettering)(Jungbluth et al. 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102(KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT WO0162931A2); and SC100 (Scancell) (PCT WO 01/88138). In another preferredembodiment, the Fc polypeptides of the present invention may find use inalemtuzumab (Campath®, Millenium), a humanized monoclonal antibodycurrently approved for treatment of B-cell chronic lymphocytic leukemia.The Fc polypeptides of the present invention may find use in a varietyof antibodies or Fc fusions that are substantially similar to otherclinical products and candidates, including but not limited tomuromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibody developed byOrtho Biotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin®), ananti-CD20 antibody developed by IDEC/Schering AG, gemtuzumab ozogamicin(Mylotarg®), an anti-CD33 (p67 protein) antibody developed byCelltech/Wyeth, alefacept (Amevive®), an anti-LFA-3 Fc fusion developedby Biogen), abciximab (ReoPro®), developed by Centocor/Lilly,basiliximab (Simulect®), developed by Novartis, palivizumab (Synagis®),developed by MedImmune, infliximab (Remicade®), an anti-TNFalphaantibody developed by Centocor, adalimumab (Humira®), an anti-TNFalphaantibody developed by Abbott, Humicade™, an anti-TNFalpha antibodydeveloped by Celltech, etanercept (Enbrel®), an anti-TNFalpha Fc fusiondeveloped by Immunex/Amgen, ABX-CBL, an anti-CD147 antibody beingdeveloped by Abgenix, ABX-IL8, an anti-IL8 antibody being developed byAbgenix, ABX-MA1, an anti-MUC18 antibody being developed by Abgenix,Pemtumomab (R1549, ⁹⁰Y-muHMFG1), an anti-MUC1 In development byAntisoma, Therex (R1550), an anti-MUC1 antibody being developed byAntisoma, AngioMab (AS1405), being developed by Antisoma, HuBC-1, beingdeveloped by Antisoma, Thioplatin (AS1407) being developed by Antisoma,Antegren® (natalizumab), an anti-alpha-4-beta-1 (VLA-4) andalpha-4-beta-7 antibody being developed by Biogen, VLA-1 mAb, ananti-VLA-1 integrin antibody being developed by Biogen, LTBR mAb, ananti-lymphotoxin beta receptor (LTBR) antibody being developed byBiogen, CAT-152, an anti-TGF-β2 antibody being developed by CambridgeAntibody Technology, J695, an anti-IL-12 antibody being developed byCambridge Antibody Technology and Abbott, CAT-192, an anti-TGFβ1antibody being developed by Cambridge Antibody Technology and Genzyme,CAT-213, an anti-Eotaxin1 antibody being developed by Cambridge AntibodyTechnology, LymphoStat-B™ an anti-Blys antibody being developed byCambridge Antibody Technology and Human Genome Sciences Inc., TRAIL-R1mAb, an anti-TRAIL-R1 antibody being developed by Cambridge AntibodyTechnology and Human Genome Sciences, Inc., Avastin™ (bevacizumab,rhuMAb-VEGF), an anti-VEGF antibody being developed by Genentech, ananti-HER receptor family antibody being developed by Genentech,Anti-Tissue Factor (ATF), an anti-Tissue Factor antibody being developedby Genentech, Xolair™ (Omalizumab), an anti-IgE antibody being developedby Genentech, Raptiva™ (Efalizumab), an anti-CD11a antibody beingdeveloped by Genentech and Xoma, MLN-02 Antibody (formerly LDP-02),being developed by Genentech and Millenium Pharmaceuticals, HuMax CD4,an anti-CD4 antibody being developed by Genmab, HuMax-IL15, an anti-IL15antibody being developed by Genmab and Amgen, HuMax-Inflam, beingdeveloped by Genmab and Medarex, HuMax-Cancer, an anti-Heparanase Iantibody being developed by Genmab and Medarex and Oxford GcoSciences,HuMax-Lymphoma, being developed by Genmab and Amgen, HuMax-TAC, beingdeveloped by Genmab, IDEC-131, and anti-CD40L antibody being developedby IDEC Pharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibodybeing developed by IDEC Pharmaceuticals, IDEC-114, an anti-CD80 antibodybeing developed by IDEC Pharmaceuticals, IDEC-152, an anti-CD23 beingdeveloped by IDEC Pharmaceuticals, anti-macrophage migration factor(MIF) antibodies being developed by IDEC Pharmaceuticals, BEC2, ananti-idiotypic antibody being developed by Imclone, IMC-1C11, ananti-KDR antibody being developed by Imclone, DC101, an anti-flk-1antibody being developed by Imclone, anti-VE cadherin antibodies beingdeveloped by Imclone, CEA-Cide™ (labetuzumab), an anti-carcinoembryonicantigen (CEA) antibody being developed by Immunomedics, LymphoCide™(Epratuzumab), an anti-CD22 antibody being developed by Immunomedics,AFP-Cide, being developed by Immunomedics, MyelomaCide, being developedby Immunomedics, LkoCide, being developed by Immunomedics, ProstaCide,being developed by Immunomedics, MDX-010, an anti-CTLA4 antibody beingdeveloped by Medarex, MDX-060, an anti-CD30 antibody being developed byMedarex, MDX-070 being developed by Medarex, MDX-018 being developed byMedarex, Osidem™ (IDM-1), and anti-Her2 antibody being developed byMedarex and Immuno-Designed Molecules, HuMax™-CD4, an anti-CD4 antibodybeing developed by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibodybeing developed by Medarex and Genmab, CNTO 148, an anti-TNFα antibodybeing developed by Medarex and Centocor/J&J, CNTO 1275, an anti-cytokineantibody being developed by Centocor/J&J, MOR101 and MOR102,anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies beingdeveloped by MorphoSys, MOR201, an anti-fibroblast growth factorreceptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion®(visilizumab), an anti-CD3 antibody being developed by Protein DesignLabs, HuZAF™, an anti-gamma interferon antibody being developed byProtein Design Labs, Anti-α5β1 Integrin, being developed by ProteinDesign Labs, anti-IL-12, being developed by Protein Design Labs, ING-1,an anti-Ep-CAM antibody being developed by Xoma, and MLN01, ananti-Beta2 integrin antibody being developed by Xoma, all referencesincorporated by reference.

Application of the Fc polypeptides to the aforementioned antibody and Fcfusion clinical products and candidates is not meant to be constrainedto their precise composition. The Fc polypeptides of the presentinvention may be incorporated into the aforementioned clinicalcandidates and products, or into antibodies and Fc fusions that aresubstantially similar to them. The Fc polypeptides of the presentinvention may be incorporated into versions of the aforementionedclinical candidates and products that are humanized, affinity matured,engineered, or modified in some other way. Furthermore, the entirepolypeptide of the aforementioned clinical products and candidates neednot be used to construct a new antibody or Fc fusion that incorporatesthe Fc polypeptides of the present invention; for example only thevariable region of a clinical product or candidate antibody, asubstantially similar variable region, or a humanized, affinity matured,engineered, or modified version of the variable region may be used. Inanother embodiment, the Fc polypeptides of the present invention mayfind use in an antibody or Fc fusion that binds to the same epitope,antigen, ligand, or receptor as one of the aforementioned clinicalproducts and candidates.

In one embodiment, the Fc polypeptides of the present invention are usedfor the treatment of autoimmune, inflammatory, or transplantindications. Target antigens and clinical products and candidates thatare relevant for such diseases include but are not limited to anti-α4β7integrin antibodies such as LDP-02, anti-beta2 integrin antibodies suchas LDP-01, anti-complement (C5) antibodies such as 5G1.1, anti-CD2antibodies such as BTI-322, MEDI-507, anti-CD3 antibodies such as OKT3,SMART anti-CD3, anti-CD4 antibodies such as IDEC-151, MDX-CD4, OKT4A,anti-CD11a antibodies, anti-CD14 antibodies such as IC14, anti-CD18antibodies, anti-CD23 antibodies such as IDEC 152, anti-CD25 antibodiessuch as Zenapax, anti-CD40L antibodies such as 5c8, Antova, IDEC-131,anti-CD64 antibodies such as MDX-33, anti-CD80 antibodies such asIDEC-114, anti-CD147 antibodies such as ABX-CBL, anti-E-selectinantibodies such as CDP850, anti-gpIIb/IIIa antibodies such asReoPro/Abcixima, anti-ICAM-3 antibodies such as ICM3, anti-ICEantibodies such as VX-740, anti-FcR1 antibodies such as MDX-33, anti-IgEantibodies such as rhuMab-E25, anti-IL-4 antibodies such as SB-240683,anti-IL-5 antibodies such as SB-240563, SCH55700, anti-IL-8 antibodiessuch as ABX-IL8, anti-interferon gamma antibodies, anti-TNF (TNF, TNFα,TNFa, TNF-alpha) antibodies such as CDP571, CDP870, D2E7, Infliximab,MAK-195F, and anti-VLA-4 antibodies such as Antegren.

Fc variants of the present invention may be utilized in TNF inhibitormolecules to provide enhanced properties. It has been shown that theeffector function associated with FcγRIIIa may negatively impact theeffectiveness of certain TNF inhibitor molecules used in the treatmentof rheumatoid arthritis or psoriatic arthritis patients that have ahigh-affinity polymorphism (158 F:V discussed herein elsewhere) andvice-versa (Z. Tutuncu et al., 2004, “FcR Polymorphisms and TreatmentOutcomes in Patients with Inflammatory Arthritis Treated with TNFBlocking Agents”, oral presentation on Oct. 18, 2004 at the 2004 ACRMeeting, San Antonio, Tex.; abstract published in Arthritis &Rheumatism, September 2004, incorporated by reference). In general forautoimmune conditions such as rheumatoid arthritis or psoriaticarthritis, combining a TNF inhibitor with an Fc variant that providesreduced binding to one or more FcγRs as compared to the parent enhancesthe effectiveness of therapy. Ideally, reduced or even ablated bindingto one or more FcγRs, for example FcγRIIIa, with a TNF inhibitormolecule would produce the best results.

Useful TNF inhibitor molecules include any molecule that inhibits theaction of TNF-alpha in a mammal. Suitable examples include the Fc fusionEnbrel® (etanercept) and the antibodies Humira® (adalimumab) andRemicade® (infliximab). Monoclonal antibodies (such as Remicade andHumira) engineered using the Fc variants of the present invention toreduce Fc binding, may translate to better efficacy. Effector functionof Humira, Remicade, and Enbrel was not considered in the development ofthese drugs, let alone modulation of effector function. By using an Fcvariant of the present invention that reduces binding to one or moreFcγRs in the context of an antibody or Fc fusion that acts on autoimmuneconditions, efficacy may be enhanced as compared to the currentlycommercialized products. Useful TNF inhibitor molecules preferablyinclude Dominant Negative TNF molecules (as defined in U.S. Ser. No.09/798,789, filed Mar. 2, 2000; U.S. Ser. No. 09/981,289, filed Oct. 15,2001; U.S. Ser. No. 10/262,630, filed Sep. 30, 2002; and U.S. Ser. No.10/963,994, filed Oct. 12, 2004, all incorporated by reference). TheDominant Negative TNF molecules (DN-TNF) have no intrinsic effectoractivity, and act to “save” transmembrane TNF (tmTNF) (i.e., if thekilling of cells that contain tmTNF has a negative effect on diseaseoutcome for rheumatoid or psoriatic arthritis). A DN-TNF moleculeassociated with an Fc variant that reduces or ablates FcγR binding tothe receptor is preferred.

In one embodiment, the Fc polypeptides of the present invention functiontherapeutically, in whole or in part, through ADCC activity. Targetantigens and clinical products and candidates that are relevant for suchapplication may include but are not limited to: anti-CD20 antibodiessuch as Bexocar, Rituxan®, Zevalin®, and PRO70769, anti-CD33 antibodiessuch as Smart M195, anti-CD22 antibodies such as Lymphocide™, anti-CD30antibodies such as AC-10 and SGN-30, anti-EGFR antibodies such asABX-EGF, Cetuximab, IMC-C225, Merck Mab 425, anti-EpCAM antibodies suchas Crucell's anti-EpCAM, anti-HER2 antibodies such as Herceptin andMDX-210, and anti-CEA antibodies such as cantumab and Pentacea.

In one embodiment, the Fc polypeptides of the present invention functiontherapeutically, in whole or in part, through CDC activity. Targetantigens and clinical products and candidates that are relevant for suchapplication may include but are not limited to: anti-CEA antibodies suchas cantumab and Pentacea, anti-CD20 antibodies such as Bexocar,Rituxan®, Zevalin®, and PRO70769, anti-EpCAM antibodies such asCrucell's anti-EpCAM and Edrecolomab, and anti-CD52 antibodies such asCampath® (alemtuzumab).

In one embodiment, the Fc polypeptides of the present invention aredirected against antigens expressed in the hematological lineage. Targetantigens and clinical products and candidates that are relevant for suchapplication may include but are not limited to: anti-CD33 antibodiessuch as Smart M195, anti-CD40L antibodies such as Antova™, IDEC-131,anti-CD44 antibodies such as Blvatuzumab, anti-CD52 antibodies such asCampath® (alemtuzumab), anti-CD80 antibodies such as IDEC-114,anti-CTLA-4 antibodies such as MDX-101, anti-CD20 antibodies such asBexocar, Rituxan®, Zevalin®, and PRO70769, anti-CD22 antibodies such asLymphocide™, anti-CD23 antibodies such as IDEC-152, anti-CD25 antibodiessuch as Zenapax® (daclizumab), and anti-MHC (HLA-DR) antibodies such asapolizumab.

In one embodiment, the Fc polypeptides of the present invention aredirected against antigens expressed in solid tumors. Target antigens andclinical products and candidates that are relevant for such applicationmay include but are not limited to: anti-EpCAM antibodies such asCrucell's anti-EpCAM and Edrecolomab, anti-CEA antibodies such ascantumab and Pentacea, anti-EGFR antibodies such as ABX-EGF, Cetuximab,IMC-C225, Merck Mab 425, anti-Mud antibodies such as BravaRex, TriAb,anti-Her2 antibodies such as Herceptin®, MDX-210, anti-GD-2 gangliosideantibodies such as 3F8 and TriGem, anti-GD-3 ganglioside antibodies suchas mitumomab, anti-PSMA antibodies such as MDX-070, anti-CA125antibodies such as oregovomab, anti-TAG-72 antibodies such as MDX-220,and anti-MUC-1 antibodies such as cantuzumab.

In a preferred embodiment, the target of the Fc variants of the presentinvention is itself one or more Fc ligands. Fc polypeptides of theinvention can be utilized to modulate the activity of the immune system,and in some cases to mimic the effects of IVIg therapy in a morecontrolled, specific, and efficient manner. IVIg is effectively a highdose of immunoglobulins delivered intravenously. In general, IVIg hasbeen used to down-regulate autoimmune conditions. It has beenhypothesized that the therapeutic mechanism of action of IVIg involvesligation of Fc receptors at high frequency (J. Bayry et al., 2003,Transfusion Clinique et Biologique 10: 165-169; Binstadt et al., 2003,J. Allergy Clin. Immunol, 697-704). Indeed animal models of(thrombocytopenia purpura (ITP) show that the isolated Fc are the activeportion of IVIg (Samuelsson et al, 2001, Pediatric Research 50(5), 551).For use in therapy, immunoglobulins are harvested from thousands ofdonors, with all of the concomitant problems associated withnon-recombinant biotherapeutics collected from humans. An Fc variant ofthe present invention should serve all of the roles of IVIg while beingmanufactured as a recombinant protein rather than harvested from donors.

The immunomodulatory effects of IVIg may be dependent on productiveinteraction with one or more Fc ligands, including but not limited toFcγRs, complement proteins, and FcRn. In some embodiments, Fc variantsof the invention with enhanced affinity for FcγRIIb can be used topromote anti-inflammatory activity (Samuelsson et al., 2001, Science291: 484-486) and or to reduce autoimmunity (Hogarth, 2002, CurrentOpinion in Immunology, 14:798-802). In other embodiments, Fcpolypeptides of the invention with enhanced affinity for one or moreFcγRs can be utilized by themselves or in combination with additionalmodifications to reduce autoimmunity (Hogarth, 2002, Current Opinion inImmunology, 14:798-802). In alternative embodiments, Fc variants of theinvention with enhanced affinity for FcγRIIIa but reduced capacity forintracellular signaling can be used to reduce immune system activationby competitively interfering with FcγRIIIa binding. The context of theFc variant dramatically impacts the desired specificity. For example, Fcvariants that provide enhanced binding to one or more activating FcγRsmay provide optimal immunomodulatory effects in the context of anantibody, Fc fusion, isolated Fc, or Fc fragment by acting as an FcγRantagonist (van Mirre et al., 2004, J. Immunol. 173:332-339). However,fusion or conjugation of two or more Fc variants may provide differenteffects, and for such an Fc polypeptide it may be optimal to utilize Fcvariants that provide enhanced affinity for an inhibitory receptor.

The Fc variants of the present invention may be used as immunomodulatorytherapeutics. Binding to or blocking Fc receptors on immune system cellsmay be used to influence immune response in immunological conditionsincluding but not limited to idiopathic thrombocytopenia purpura (ITP)and rheumatoid arthritis (RA) among others. By use of the affinityenhanced Fc variants of the present invention, the dosages required intypical IVIg applications may be reduced while obtaining a substantiallysimilar therapeutic effect. The Fc variants may provide enhanced bindingto an FcγR, including but not limited to FcγRIIa, FcγRIIb, FcγRIIIa,FcγRIIIb, and/or FcγRI. In particular, binding enhancements to FcγRIIbwould increase expression or inhibitory activity, as needed, of thatreceptor and improve efficacy. Alternatively, blocking binding toactivation receptors such as FcγRIIIb or FcγRI may improve efficacy. Inaddition, modulated affinity of the Fc variants for FcRn and/or alsocomplement may also provide benefits.

In one embodiment, Fc variants that provide enhanced binding to theinhibitory receptor FcγRIIb provide an enhancement to the IVIgtherapeutic approach. In particular, the Fc variants of the presentinvention that bind with greater affinity to the FcγRIIb receptor thanparent Fc polypeptide may be used. Such Fc variants would thus functionas FcγRIIb agonists, and would be expected to enhance the beneficialeffects of IVIg as an autoimmune disease therapeutic and also as amodulator of B-cell proliferation. In addition, such FcγRIIb-enhanced Fcvariants may also be further modified to have the same or limitedbinding to other receptors. In additional embodiments, the Fc variantswith enhanced FcγRIIb affinity may be combined with mutations thatreduce or ablate to other receptors, thereby potentially furtherminimizing side effects during therapeutic use.

Such immunomodulatory applications of the Fc variants of the presentinvention may also be utilized in the treatment of oncologicalindications, especially those for which antibody therapy involvesantibody-dependant cytotoxic mechanisms. For example, an Fc variant thatenhances affinity to FcγRIIb may be used to antagonize this inhibitoryreceptor, for example by binding to the Fc/FcγRIIb binding site butfailing to trigger, or reducing cell signaling, potentially enhancingthe effect of antibody-based anti-cancer therapy. Such Fc variants,functioning as FcγRIIb antagonists, may either block the inhibitoryproperties of FcγRIIb, or induce its inhibitory function as in the caseof IVIg. An FcγRIIb antagonist may be used as co-therapy in combinationwith any other therapeutic, including but not limited to antibodies,acting on the basis of ADCC related cytotoxicity. FcγRIIb antagonisticFc variants of this type are preferably isolated Fc or Fc fragments,although in alternate embodiments antibodies and Fc fusions may be used.

Optimized Properties

The present invention provides Fc variants that are optimized for anumber of therapeutically relevant properties. An Fc variant comprisesone or more amino acid modifications relative to a parent Fcpolypeptide, wherein said amino acid modification(s) provide one or moreoptimized properties. An Fc variant of the present invention differs inamino acid sequence from its parent Fc polypeptide by virtue of at leastone amino acid modification. Thus Fc variants of the present inventionhave at least one amino acid modification compared to the parent.Alternatively, the Fc variants of the present invention may have morethan one amino acid modification as compared to the parent, for examplefrom about one to fifty amino acid modifications, preferrably from aboutone to ten amino acid modifications, and most preferably from about oneto about five amino acid modifications compared to the parent. Thus thesequences of the Fc variants and those of the parent Fc polypeptide aresubstantially homologous. For example, the variant Fc variant sequencesherein will possess about 80% homology with the parent Fc variantsequence, preferably at least about 90% homology, and most preferably atleast about 95% homology.

The Fc variants of the present invention may be optimized for a varietyof properties. An Fc variant that is engineered or predicted to displayone or more optimized properties is herein referred to as an “optimizedFc variant”. Properties that may be optimized include but are notlimited to enhanced or reduced affinity for an FcγR. In a preferredembodiment, the Fc variants of the present invention are optimized topossess enhanced affinity for a human activating FcγR, preferably FcγRI,FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb, most preferably FcγRIIIa. Inan alternately preferred embodiment, the Fc variants are optimized topossess reduced affinity for the human inhibitory receptor FcγRIIb.These preferred embodiments are anticipated to provide Fc polypeptideswith enhanced therapeutic properties in humans, for example enhancedeffector function and greater anti-cancer potency. In an alternateembodiment, the Fc variants of the present invention are optimized tohave reduced or ablated affinity for a human FcγR, including but notlimited to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb.These embodiments are anticipated to provide Fc polypeptides withenhanced therapeutic properties in humans, for example reduced effectorfunction and reduced toxicity. In other embodiments, Fc variants of thepresent invention provide enhanced affinity for one or more FcγRs, yetreduced affinity for one or more other FcγRs. For example, an Fc variantof the present invention may have enhanced binding to FcγRIIIa, yetreduced binding to FcγRIIb. Alternately, an Fc variant of the presentinvention may have enhanced binding to FcγRIIa and FcγRI, yet reducedbinding to FcγRIIb. In yet another embodiment, an Fc variant of thepresent invention may have enhanced affinity for FcγRIIb, yet reducedaffinity to one or more activating FcγRs.

Preferred embodiments comprise optimization of Fc binding to a humanFcγR, however in alternate embodiments the Fc variants of the presentinvention possess enhanced or reduced affinity for FcγRs from nonhumanorganisms, including but not limited to rodents and non-human primates.Fc variants that are optimized for binding to a nonhuman FcγR may finduse in experimentation. For example, mouse models are available for avariety of diseases that enable testing of properties such as efficacy,toxicity, and pharmacokinetics for a given drug candidate. As is knownin the art, cancer cells can be grafted or injected into mice to mimic ahuman cancer, a process referred to as xenografting. Testing of Fcvariants that comprise Fc variants that are optimized for one or moremouse FcγRs, may provide valuable information with regard to theefficacy of the protein, its mechanism of action, and the like. The Fcvariants of the present invention may also be optimized for enhancedfunctionality and/or solution properties in aglycosylated form. In apreferred embodiment, the aglycosylated Fc variants of the presentinvention bind an Fc ligand with greater affinity than the aglycosylatedform of the parent Fc variant. Said Fc ligands include but are notlimited to FcγRs, C1q, FcRn, and proteins A and G, and may be from anysource including but not limited to human, mouse, rat, rabbit, ormonkey, preferably human. In an alternately preferred embodiment, the Fcvariants are optimized to be more stable and/or more soluble than theaglycosylated form of the parent Fc variant.

Fc variants of the invention may comprise modifications that modulateinteraction with Fc ligands other than FcγRs, including but not limitedto complement proteins, FcRn, and Fc receptor homologs (FcRHs). FcRHsinclude but are not limited to FcRH1, FcRH2, FcRH3, FcRH4, FcRH5, andFcRH6 (Davis et al., 2002, Immunol. Reviews 190:123-136).

Preferably, the Fc ligand specificity of the Fc variant of the presentinvention will determine its therapeutic utility. The utility of a givenFc variant for therapeutic purposes will depend on the epitope or formof the Target antigen and the disease or indication being treated. Forsome targets and indications, enhanced FcγR-mediated effector functionsmay be preferable. This may be particularly favorable for anti-cancer Fcvariants. Thus Fc variants may be used that comprise Fc variants thatprovide enhanced affinity for activating FcγRs and/or reduced affinityfor inhibitory FcγRs. For some targets and indications, it may befurther beneficial to utilize Fc variants that provide differentialselectivity for different activating FcγRs; for example, in some casesenhanced binding to FcγRIIa and FcγRIIIa may be desired, but not FcγRI,whereas in other cases, enhanced binding only to FcγRIIa may bepreferred. For certain targets and indications, it may be preferable toutilize Fc variants that enhance both FcγR-mediated andcomplement-mediated effector functions, whereas for other cases it maybe advantageous to utilize Fc variants that enhance either FcγR-mediatedor complement-mediated effector functions. For some Targets or cancerindications, it may be advantageous to reduce or ablate one or moreeffector functions, for example by knocking out binding to C1q, one ormore FcγR's, FcRn, or one or more other Fc ligands. For other targetsand indications, it may be preferable to utilize Fc variants thatprovide enhanced binding to the inhibitory FcγRIIb, yet WT level,reduced, or ablated binding to activating FcγRs. This may beparticularly useful, for example, when the goal of an Fc variant is toinhibit inflammation or auto-immune disease, or modulate the immunesystem in some way.

Clearly an important parameter that determines the most beneficialselectivity of a given Fc variant to treat a given disease is thecontext of the Fc variant, that is what type of Fc variant is beingused. Thus the Fc ligand selectivity or specificity of a given Fcvariant will provide different properties depending on whether itcomposes an antibody, Fc fusion, or an Fc variants with a coupled fusionor conjugate partner. For example, toxin, radionucleotide, or otherconjugates may be less toxic to normal cells if the Fc variant thatcomprises them has reduced or ablated binding to one or more Fc ligands.As another example, in order to inhibit inflammation or auto-immunedisease, it may be preferable to utilize an Fc variant with enhancedaffinity for activating FcγRs, such as to bind these FcγRs and preventtheir activation. Conversely, an Fc variant that comprises two or moreFc regions with enhanced FcγRIIb affinity may co-engage this receptor onthe surface of immune cells, thereby inhibiting proliferation of thesecells. Whereas in some cases an Fc variants may engage its targetantigen on one cell type yet engage FcγRs on separate cells from thetarget antigen, in other cases it may be advantageous to engage FcγRs onthe surface of the same cells as the target antigen. For example, if anantibody targets an antigen on a cell that also expresses one or moreFcγRs, it may be beneficial to utilize an Fc variant that enhances orreduces binding to the FcγRs on the surface of that cell. This may bethe case, for example when the Fc variant is being used as ananti-cancer agent, and co-engagement of target antigen and FcγR on thesurface of the same cell promote signaling events within the cell thatresult in growth inhibition, apoptosis, or other anti-proliferativeeffect. Alternatively, antigen and FcγR co-engagement on the same cellmay be advantageous when the Fc variant is being used to modulate theimmune system in some way, wherein co-engagement of target antigen andFcγR provides some proliferative or anti-proliferative effect. Likewise,Fc variants that comprise two or more Fc regions may benefit from Fcvariants that modulate FcγR selectivity or specificity to co-engageFcγRs on the surface of the same cell.

The Fc ligand specificity of the Fc variants of the present inventioncan be modulated to create different effector function profiles that maybe suited for particular target antigens, indications, or patientpopulations. Table 1 describes several preferred embodiments of receptorbinding profiles that include improvements to, reductions to or noeffect to the binding to various receptors, where such changes may bebeneficial in certain contexts. The receptor binding profiles in thetable could be varied by degree of increase or decrease to the specifiedreceptors. Additionally, the binding changes specified could be in thecontext of additional binding changes to other receptors such as C1q orFcRn, for example by combining with ablation of binding to C1q to shutoff complement activation, or by combining with enhanced binding to C1qto increase complement activation. Other embodiments with other receptorbinding profiles are possible, the listed receptor binding profiles areexemplary.

TABLE 1 Affinity Affinity Enhancement Reduction Cell ActivityTherapeutic Activity FcγRI only — Enhanced dendritic cell activityEnhanced cell-based and uptake, and subsequent immune response againstpresentation of antigens target Enhanced monocyte and macrophageresponse to antibody FcγRIIIa — Enhanced ADCC and Increased target celllysis phagocytosis of broad range of cell types FcγRIIIa FcγRIIbEnhanced ADCC and Increased target cell lysis phagocytosis of broadrange of cell types FcγRIIb — Reduced activity of all FcγR Enhancementof target cell FcγRIIc bearing cell types except NK cells lysisselective for NK cell Possible activation of NK cells accessible targetcells via FcγRIIc receptor signaling FcγRIIb — Possible NK cell specificEnhanced target cell lysis FcγRIIIa activation and enhancement ofselective for NK cell NK cell mediated ADCC accessible target cellsFcγRIIIb — Neutrophil mediated Enhanced target cell phagocytosisenhancement destruction for neutrophil accessible cells FcαR —Neutrophil mediated Enhanced target cell phagocytosis enhancementdestruction for neutrophil accessible cells FcγRI FcγRIIb Enhanceddendritic cell activity Enhanced cell-based FcγRIIa and uptake, andsubsequent immune response against FcγRIIIa presentation of antigens toT cells target Enhanced monocyte and macrophage response to antibodyFcγRIIb FcγRI Reduced activity of monocytes, Eliminated or reduced cell-FcγRIIa macrophages, neutrophils, NK, mediated cytotoxicity FcγRIIIadendritic and other gamma against target bearing cells receptor bearingcells

The presence of different polymorphic forms of FcγRs provides yetanother parameter that impacts the therapeutic utility of the Fcvariants of the present invention. Whereas the specificity andselectivity of a given Fc variant for the different classes of FcγRssignificantly affects the capacity of an Fc variant to target a givenantigen for treatment of a given disease, the specificity or selectivityof an Fc variant for different polymorphic forms of these receptors mayin part determine which research or pre-clinical experiments may beappropriate for testing, and ultimately which patient populations may ormay not respond to treatment. Thus the specificity or selectivity of Fcvariants of the present invention to Fc ligand polymorphisms, includingbut not limited to FcγR, C1q, FcRn, and FcRH polymorphisms, may be usedto guide the selection of valid research and pre-clinical experiments,clinical trial design, patient selection, dosing dependence, and/orother aspects concerning clinical trials.

Additional Modifications

In addition to comprising an Fc variant of the present invention, the Fcpolypeptides of the present invention may comprise one or moreadditional modifications. Said modifications may be amino acidmodifications, or may modifications that are not amino acidmodifications such as modifications that are made enzymatically orchemically. Combinations of additional amino acid modifications andmodifications that are not amino acid modifications are contemplated.Such additional modification(s) likely provide some improvement in theFc polypeptide, for example an enhancement in its stability, solubility,function, or clinical use. The present invention contemplates a varietyof improvements that made be made by coupling the Fc variants of thepresent invention with additional modifications.

The Fc variants of the present invention may be combined with otheramino acid modifications in the Fc region that provide altered oroptimized interaction with one or more Fc ligands, including but notlimited to FcγRs, C1q or other complement proteins, FcRn, FcR homologues(FcRHs), and/or as yet undiscovered Fc ligands. It is noted that Fcpolypeptides of the present invention may themselves have as yet unknownuseful interaction properties with one or more Fc ligands, for exampleFcRHs. Additional modifications may provide altered or optimizedaffinity and/or specificity to the Fc ligands. Additional modificationsmay provide altered or optimized effector functions, including but notlimited to ADCC, ADCP, CDC, and/or serum half-life. Such combination mayprovide additive, synergistic, or novel properties. In one embodiment,the Fc variants of the present invention may be combined with known Fcvariants (Duncan et al., 1988, Nature 332:563-564; Lund et al., 1991, JImmunol 147:2657-2662; Lund et al., 1992, Mol Immunol 29:53-59; Alegreet al., 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, ProcNatl Acad Sci USA 92:11980-11984; Jefferis et al., 1995, Immunol Lett44:111-117; Lund et al., 1995, Faseb J9:115-119; Jefferis et al., 1996,Immunol Lett 54:101-104; Lund et al., 1996, J Immunol 157:4963-4969;Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al., 2000,J Immunol 164:4178-4184; Reddy et al., 2000, J Immunol 164:1925-1933; Xuet al., 2000, Cell Immunol 200:16-26; Idusogie et al., 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al., 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem SocTrans 30:487-490; Hinton et al., 2004, J Biol Chem 279:6213-6216) (U.S.Pat. No. 5,624,821; U.S. Pat. No. 5,885,573; U.S. Pat. No. 6,194,551;PCT WO 00/42072; PCT WO 99/58572; US 2004/0002587 Al), U.S. Pat. No.6,737,056, PCT US2004/000643, U.S. Ser. No. 10/370,749, andPCT/US2004/005112), all incorporated by reference. For example, asdescribed in U.S. Pat. No. 6,737,056, PCT US2004/000643, U.S. Ser. No.10/370,749, and PCT/US2004/005112, the substitutions S298A, S298D,K326E, K326D, E333A, K334A, and P396L provide optimized FcγR bindingand/or enhanced ADCC. Furthermore, as disclosed in Idusogie et al.,2001, J. Immunology 166:2571-2572, incorporated by reference,substitutions K326W, K326Y, and E333S provide enhanced binding to thecomplement protein C1q and enhanced CDC. Finally, as described in Hintonet al., 2004, J. Biol. Chem. 279(8): 6213-6216, incorporated byreference, substitutions T250Q, T250E, M428L, and M428F provide enhancedbinding to FcRn and improved pharmacokinetics.

Because the binding sites for FcγRs, C1q, and FcRn reside in the Fcregion, the differences between the IgGs in the Fc region are likely tocontribute to differences in FcγR- and C1q-mediated effector functions.It is also possible that the modifications can be made in other non-Fcregions of an Fc variant, including for example the Fab and hingeregions of an antibody, or the Fc fusion partner of an Fc fusion. Forexample, as disclosed in U.S. Ser. No. 11/090,981, hereby incorporatedby reference, the Fab and hinge regions of an antibody may impacteffector functions such as antibody dependent cell-mediated cytotoxicity(ADCC), antibody dependent cell-mediated phagocytosis (ADCP), andcomplement dependent cytotoxicity (CDC). Thus modifications outside theFc region of an Fc variant of the present invention are contemplated.For example, antibodies of the present invention may comprise one ormore amino acid modifications in the V_(L), C_(L), V_(H), C_(H)1, and/orhinge regions of an antibody.

Other modifications may provide additional or novel binding determinantsinto an Fc variant, for example additional or novel Fc receptor bindingsites, for example as described in U.S. Ser. No. 60/531,752, filed Dec.12, 2003, entitled “Fc variants with novel Fc receptor binding sites”.In one embodiment, an Fc variant of one antibody isotype may beengineered such that it binds to an Fc receptor of a different isotype.This may be particularly applicable when the Fc binding sites for therespective Fc receptors do not significantly overlap. For example, thestructural determinants of IgA binding to FcγRI may be engineered intoan IgG Fc variant.

The Fc variants of the present invention may comprise modifications thatmodulate the in vivo pharmacokinetic properties of an Fc variant. Theseinclude, but are not limited to, modifications that enhance affinity forthe neonatal Fc receptor FcRn (U.S. Ser. No. 10/020,354;WO2001US0048432; EP2001000997063; U.S. Pat. No. 6,277,375; U.S. Ser. No.09/933,497; WO1997US0003321; U.S. Pat. No. 6,737,056; WO2000US0000973;Shields et al., 2001, J Biol Chem 276(9): 6591-6604; Zhou et al., 2003,J Mol. Biol., 332: 901-913). These further include modifications thatmodify FcRn affinity in a pH-specific manner. In some embodiments, whereenhanced in vivo half-life is desired, modifications that specificallyenhance FcRn affinity at lower pH (5.5-6) relative to higher pH (7-8)are preferred (Hinton et al., 2004, J Biol Chem 279(8): 6213-6216; Dall'Acqua et al., 2002 J Immuno 169: 5171-5180; Ghetie et al., 1997, NatBiotechnol 15(7): 637-640; WO2003US0033037; WO2004US0011213). Forexample, as described in Hinton et al., 2004, “Engineered Human IgGAntibodies with Longer Serum Half-lives in Primates” J Biol Chem 279(8):6213-6216, substitutions T250Q, T250E, M428L, and M428F provide enhancedbinding to FcRn and improved pharmacokinetics. Additionally preferredmodifications are those that maintain the wild-type Fc's improvedbinding at lower pH relative to the higher pH. In alternativeembodiments, where rapid in vivo clearance is desired, modificationsthat reduce affinity for FcRn are preferred. (U.S. Pat. No. 6,165,745;W01993U50003895; EP1993000910800; W01997U50021437; Medesan et al., 1997,J Immunol 158(5): 2211-2217; Ghetie & Ward, 2000, Annu Rev Immunol 18:739-766; Martin et al. 2001, Molecular Cell 7: 867-877; Kim et al. 1999,Eur J Immunol 29: 2819-2825). Preferred variants that enhance FcRn aredescribed in U.S. Ser. No. 60/627,763, filed Nov. 12, 2004; 60/642,886,filed Jan. 11, 2005; 60/649,508, filed Feb. 2, 2005; 60/662,468, filedMar. 15, 2005, and 60/669,311 filed Apr. 6, 2005, entitled “Fc Variantswith Altered Binding to FcRn”, all hereby incorporated by reference.

Additional modifications may comprise amino acid modifications whereinresidues in an Fc polypeptide are modified to the corresponding residuein a homologous Fc polypeptide. Effector functions such as ADCC, ADCP,CDC, and serum half-life differ significantly between the differentclasses of antibodies, including for example human IgG1, IgG2, IgG3,IgG4, IgA1, IgA2, IgD, IgE, IgG, and IgM (references—Michaelsen et al.,1992, Molecular Immunology 29(3): 319-326). Human IgG1 is the mostcommonly used antibody for therapeutic purposes, and engineering studieswherein variants have been constructed that show enhanced effectorfunction have been carried out predominantly in this context. Asdescribed above, it is possible to determine corresponding or equivalentresidues in Fc polypeptides that have significant sequence or structuralhomology with each other. By the same token, it is possible to use suchmethods to engineer additional amino acid modifications in an Fcpolypeptide to provide additional optimized properties, for example asdescribed in U.S. Ser. No. 60/621,387, filed Oct. 21, 2004. In oneembodiment, amino acid modifications can be made that replace one ormore residues in an Fc polypeptide of the present invention with one ormore residues in another homologous Fc polypeptide. In an alternateembodiment, hybrid Fc polypeptides are constructed, such that one ormore regions of an Fc polypeptide of the present invention are replacewith the corresponding regions of a homologus Fc polypeptide. Forexample, some studies have explored IgG1, IgG2, IgG3, and IgG4 variantsin order to investigate the determinants of the effector functiondifferences between them. See for example Canfield & Morrison, 1991, JExp Med 173: 1483-1491; Chappel et al., 1991, Proc Natl Acad Sci USA88(20): 9036-9040; Chappel et al., 1993, J Biol Chem 268: 25124-25131;Tao, Canfield, and Morrison, 1991, J Exp Med 173: 1025-1028; Tao et al.,1993, J Exp Med 178: 661-667; Redpath et al., 1998, Human Immunology,59, 720-727.

In one embodiment, the Fc variants of the present invention comprise oneor more engineered glycoforms. By “engineered glycoform” as used hereinis meant a carbohydrate composition that is covalently attached to an Fcvariant, wherein said carbohydrate composition differs chemically fromthat of a parent Fc variant. Engineered glycoforms may be useful for avariety of purposes, including but not limited to enhancing or reducingeffector function. Engineered glycoforms may be generated by a varietyof methods known in the art (Umañ a et al., 1999, Nat Biotechnol17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shieldset al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J BiolChem 278:3466-3473); (U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370;U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO02/31140A1; PCT WO 02/30954A1); (Potelligent™ technology [Biowa, Inc.,Princeton, N.J.]; GlycoMAb™ glycosylation engineering technology[GLYCART biotechnology AG, Zürich, Switzerland]). Many of thesetechniques are based on controlling the level of fucosylated and/orbisecting oligosaccharides that are covalently attached to the Fcregion, for example by expressing an Fc variant in various organisms orcell lines, engineered or otherwise (for example Lec-13 CHO cells or rathybridoma YB2/0 cells), by regulating enzymes involved in theglycosylation pathway (for example FUT8 [α1,6-fucosyltranserase] and/orβ1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by modifyingcarbohydrate(s) after the Fc variant has been expressed. Engineeredglycoform typically refers to the different carbohydrate oroligosaccharide; thus an Fc variant, for example an antibody or Fcfusion, may comprise an engineered glycoform. Alternatively, engineeredglycoform may refer to the Fc variant that comprises the differentcarbohydrate or oligosaccharide.

Fc variants of the present invention may comprise one or moremodifications that provide optimized properties that are notspecifically related to effector function per se. Said modifications maybe amino acid modifications, or may be modifications that are madeenzymatically or chemically. Such modification(s) likely provide someimprovement in the Fc variant, for example an enhancement in itsstability, solubility, function, or clinical use. The present inventioncontemplates a variety of improvements that made be made by coupling theFc variants of the present invention with additional modifications.

In a preferred embodiment, the Fc variants of the present invention maycomprise modifications to reduce immunogenicity in humans. In a mostpreferred embodiment, the immunogenicity of an Fc variant of the presentinvention is reduced using a method described in U.S. Ser. No.11/004,590, filed Dec. 3, 2004, entitled “Methods of Generating VariantProteins with Increased Host String Content and Compositions Thereof”.In alternate embodiments, the antibodies of the present invention arehumanized (Clark, 2000, Immunol Today 21:397-402). By “humanized”antibody as used herein is meant an antibody comprising a humanframework region (FR) and one or more complementarity determiningregions (CDR's) from a non-human (usually mouse or rat) antibody. Thenon-human antibody providing the CDR's is called the “donor” and thehuman immunoglobulin providing the framework is called the “acceptor”.Humanization relies principally on the grafting of donor CDRs ontoacceptor (human) V_(L) and V_(H) frameworks (Winter U.S. Pat. No.5,225,539). This strategy is referred to as “CDR grafting”.“Backmutation” of selected acceptor framework residues to thecorresponding donor residues is often required to regain affinity thatis lost in the initial grafted construct (U.S. Pat. No. 5,530,101; U.S.Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762;U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat. No.5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213). Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region, typically that of a humanimmunoglobulin, and thus will typically comprise a human Fc region. Avariety of techniques and methods for humanizing and reshaping non-humanantibodies are well known in the art (See Tsurushita & Vasquez, 2004,Humanization of Monoclonal Antibodies, Molecular Biology of B Cells,533-545, Elsevier Science (USA), and references cited therein).Humanization methods include but are not limited to methods described inJones et al., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen etal., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, JImmunol 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA89:4285-9, Presta et al., 1997, Cancer Res 57(20):4593-9; Gorman et al.,1991, Proc Natl Acad Sci USA 88:4181-4185; O'Connor et al., 1998,Protein Eng 11:321-8. Humanization or other methods of reducing theimmunogenicity of nonhuman antibody variable regions may includeresurfacing methods, as described for example in Roguska et al., 1994,Proc Natl Acad Sci USA 91:969-973. In one embodiment, selection basedmethods may be employed to humanize and/or affinity mature antibodyvariable regions, including but not limited to methods described in Wuet al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol.Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37):22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95:8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759.Other humanization methods may involve the grafting of only parts of theCDRs, including but not limited to methods described in U.S. Ser. No.09/810,502; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis etal., 2002, J. Immunol. 169:3076-3084. Structure-based methods may beemployed for humanization and affinity maturation, for example asdescribed in U.S. Ser. No. 10/153,159 and related applications.

Modifications to reduce immunogenicity may include modifications thatreduce binding of processed peptides derived from the parent sequence toMHC proteins. For example, amino acid modifications would be engineeredsuch that there are no or a minimal number of immune epitopes that arepredicted to bind, with high affinity, to any prevalent MHC alleles.Several methods of identifying MHC-binding epitopes in protein sequencesare known in the art and may be used to score epitopes in an Fc variantof the present invention. See for example WO 98/52976; WO 02/079232; WO00/3317; U.S. Ser. No. 09/903,378; U.S. Ser. No. 10/039,170; U.S. Ser.No. 60/222,697; U.S. Ser. No. 10/339,788; PCT WO 01/21823; and PCT WO02/00165; Mallios, 1999, Bioinformatics 15: 432-439; Mallios, 2001,Bioinformatics 17: 942-948; Sturniolo et al., 1999, Nature Biotech. 17:555-561; WO 98/59244; WO 02/069232; WO 02/77187; Marshall et al., 1995,J. Immunol. 154: 5927-5933; and Hammer et al., 1994, J. Exp. Med. 180:2353-2358. Sequence-based information can be used to determine a bindingscore for a given peptide—MHC interaction (see for example Mallios,1999, Bioinformatics 15: 432-439; Mallios, 2001, Bioinformatics 17:p942-948; Sturniolo et. al., 1999, Nature Biotech. 17: 555-561). It ispossible to use structure-based methods in which a given peptide iscomputationally placed in the peptide-binding groove of a given MHCmolecule and the interaction energy is determined (for example, see WO98/59244 and WO 02/069232). Such methods may be referred to as“threading” methods. Alternatively, purely experimental methods can beused; for example a set of overlapping peptides derived from the proteinof interest can be experimentally tested for the ability to induceT-cell activation and/or other aspects of an immune response. (see forexample WO 02/77187). In a preferred embodiment, MHC-binding propensityscores are calculated for each 9-residue frame along the proteinsequence using a matrix method (see Sturniolo et. al., supra; Marshallet. al., 1995, J. Immunol. 154: 5927-5933, and Hammer et. al., 1994, J.Exp. Med. 180: 2353-2358). It is also possible to consider scores foronly a subset of these residues, or to consider also the identities ofthe peptide residues before and after the 9-residue frame of interest.The matrix comprises binding scores for specific amino acids interactingwith the peptide binding pockets in different human class II MHCmolecule. In the most preferred embodiment, the scores in the matrix areobtained from experimental peptide binding studies. In an alternatepreferred embodiment, scores for a given amino acid binding to a givenpocket are extrapolated from experimentally characterized alleles toadditional alleles with identical or similar residues lining thatpocket. Matrices that are produced by extrapolation are referred to as“virtual matrices”. In an alternate embodiment, additional amino acidmodifications may be engineered to reduce the propensity of the intactmolecule to interact with B cell receptors and circulating antibodies.

Antibodies and Fc fusions of the present invention may comprise aminoacid modifications in one or more regions outside the Fc region, forexample the antibody Fab region or the Fc fusion partner, that provideoptimal properties. In one embodiment, the variable region of anantibody of the present invention may be affinity matured, that is tosay that amino acid modifications have been made in the V_(H) and/orV_(L) domains of the antibody to enhance binding of the antibody to itstarget antigen. Likewise, modifications may be made in the Fc fusionpartner to enhance affinity of the Fc fusion for its target antigen.Such types of modifications may improve the association and/or thedissociation kinetics for binding to the target antigen. Othermodifications include those that improve selectivity for target antigenvs. alternative targets. These include modifications that improveselectivity for antigen expressed on target vs. non-target cells. Otherimprovements to the target recognition properties may be provided byadditional modifications. Such properties may include, but are notlimited to, specific kinetic properties (i.e. association anddissociation kinetics), selectivity for the particular target versusalternative targets, and selectivity for a specific form of targetversus alternative forms. Examples include full-length versus splicevariants, aberrant forms of antigens that are expressed only on certaincell types such as tumor cells, cell-surface vs. soluble forms,selectivity for various polymorphic variants, or selectivity forspecific conformational forms of the target.

Fc variants of the invention may comprise one or more modifications thatprovide reduced or enhanced internalization of an Fc variant. In oneembodiment, Fc variants of the present invention can be utilized orcombined with additional modifications in order to reduce the cellularinternalization of an Fc variant that occurs via interaction with one ormore Fc ligands. This property might be expected to enhance effectorfunction, and potentially reduce immunogenicity of the Fc variants ofthe invention. Alternatively, Fc variants of the present Fc variants ofthe present invention can be utilized directly or combined withadditional modifications in order to enhance the cellularinternalization of an Fc variant that occurs via interaction with one ormore Fc ligands. For example, in a preferred embodiment, an Fc variantis used that provides enhanced binding to FcγRI, which is expressed ondendritic cells and active early in immune response. This strategy couldbe further enhanced by combination with additional modifications, eitherwithin the Fc variant or in an attached fusion or conjugate partner,that promote recognition and presentation of Fc peptide fragments by MHCmolecules. These strategies are expected to enhance target antigenprocessing and thereby improve antigenicity of the target antigen(Bonnerot and Amigorena, 1999, Immunol Rev. 172:279-84), promoting anadaptive immune response and greater target cell killing by the humanimmune system. These strategies may be particularly advantageous whenthe targeted antigen is shed from the cellular surface. An additionalapplication of these concepts arises with idiotype vaccineimmunotherapies, in which clone-specific antibodies produced by apatient's lymphoma cells are used to vaccinate the patient.

In a preferred embodiment, modifications are made to improve biophysicalproperties of the Fc variants of the present invention, including butnot limited to stability, solubility, and oligomeric state.Modifications can include, for example, substitutions that provide morefavorable intramolecular interactions in the Fc variant such as toprovide greater stability, or substitution of exposed nonpolar aminoacids with polar amino acids for higher solubility. A number ofoptimization goals and methods are described in U.S. Ser. No. 10/379,392that may find use for engineering additional modifications to furtheroptimize the Fc variants of the present invention. The Fc variants ofthe present invention can also be combined with additional modificationsthat reduce oligomeric state or size, such that tumor penetration isenhanced, or in vivo clearance rates are increased as desired.

Other modifications to the Fc variants of the present invention includethose that enable the specific formation or homodimeric orhomomultimeric molecules. Such modifications include but are not limitedto engineered disulfides, as well as chemical modifications oraggregation methods which may provide a mechanism for generatingcovalent homodimeric or homomultimers. For example, methods ofengineering and compositions of such molecules are described in Kan etal., 2001, J. Immunol., 2001, 166: 1320-1326; Stevenson et al., 2002,Recent Results Cancer Res. 159: 104-12; U.S. Pat. No. 5,681,566; Caronet al., 1992, J Exp Med 176:1191-1195, and Shopes, 1992, J Immunol148(9):2918-22. Additional modifications to the variants of the presentinvention include those that enable the specific formation orheterodimeric, heteromultimeric, bifunctional, and/or multifunctionalmolecules. Such modifications include, but are not limited to, one ormore amino acid substitutions in the C_(H)3 domain, in which thesubstitutions reduce homodimer formation and increase heterodimerformation. For example, methods of engineering and compositions of suchmolecules are described in Atwell et al., 1997, J Mol Biol 270(1):26-35,and Carter et al., 2001, J Immunol Methods 248:7-15. Additionalmodifications include modifications in the hinge and CH3 domains, inwhich the modifications reduce the propensity to form dimers.

In further embodiments, the Fc variants of the present inventioncomprise modifications that remove proteolytic degradation sites. Thesemay include, for example, protease sites that reduce production yields,as well as protease sites that degrade the administered protein in vivo.In a preferred embodiment, additional modifications are made to removecovalent degradation sites such as deamidation (i.e. deamidation ofglutaminyl and asparaginyl residues to the corresponding glutamyl andaspartyl residues), oxidation, and proteolytic degradation sites.Deamidation sites that are particular useful to remove are those thathave enhance propensity for deamidation, including, but not limited toasparaginyl and glutamyl residues followed by glycines (NG and QGmotifs, respectively). In such cases, substitution of either residue cansignificantly reduce the tendency for deamidation. Common oxidationsites include methionine and cysteine residues. Other covalentmodifications, that can either be introduced or removed, includehydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl or threonyl residues, methylation of the “-amino groups oflysine, arginine, and histidine side chains [T. E. Creighton, Proteins:Structure and Molecular Properties, W.H. Freeman & Co., San Francisco,pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation ofany C-terminal carboxyl group. Additional modifications also may includebut are not limited to posttranslational modifications such as N-linkedor O-linked glycosylation and phosphorylation.

Modifications may include those that improve expression and/orpurification yields from hosts or host cells commonly used forproduction of biologics. These include, but are not limited to variousmammalian cell lines (e.g. CHO), yeast cell lines, bacterial cell lines,and plants. Additional modifications include modifications that removeor reduce the ability of heavy chains to form inter-chain disulfidelinkages. Additional modifications include modifications that remove orreduce the ability of heavy chains to form intra-chain disulfidelinkages.

The Fc variants of the present invention may comprise modifications thatinclude the use of unnatural amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al.,2003, Science 301(5635):964-7. In some embodiments, these modificationsenable manipulation of various functional, biophysical, immunological,or manufacturing properties discussed above. In additional embodiments,these modifications enable additional chemical modification for otherpurposes. Other modifications are contemplated herein. For example, theFc variant may be linked to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol (PEG), polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol. Additional amino acid modifications may be made to enablespecific or non-specific chemical or posttranslational modification ofthe Fc variants. Such modifications, include, but are not limited toPEGylation and glycosylation. Specific substitutions that can beutilized to enable PEGylation include, but are not limited to,introduction of novel cysteine residues or unnatural amino acids suchthat efficient and specific coupling chemistries can be used to attach aPEG or otherwise polymeric moiety. Introduction of specificglycosylation sites can be achieved by introducing novel N-X-T/Ssequences into the Fc variants of the present invention.

The Fc variants of the present invention may be fused or conjugated toone or more other molecules or polypeptides. Conjugate and fusionpartners may be any molecule, including small molecule chemicalcompounds and polypeptides. For example, a variety of antibodyconjugates and methods are described in Trail et al., 1999, Curr. Opin.Immunol. 11:584-588. Possible conjugate partners include but are notlimited to cytokines, cytotoxic agents, toxins, radioisotopes,chemotherapeutic agent, anti-angiogenic agents, a tyrosine kinaseinhibitors, and other therapeutically active agents. In someembodiments, conjugate partners may be thought of more as payloads, thatis to say that the goal of a conjugate is targeted delivery of theconjugate partner to a targeted cell, for example a cancer cell orimmune cell, by the Fc variant. Thus, for example, the conjugation of atoxin to an antibody or Fc fusion targets the delivery of said toxin tocells expressing the target antigen.

In one embodiment, the Fc variants of the present invention are fused orconjugated to a cytokine. By “cytokine” as used herein is meant ageneric term for proteins released by one cell population that act onanother cell as intercellular mediators. For example, as described inPenichet et al., 2001, J Immunol Methods 248:91-101, cytokines may befused to antibody to provide an array of desirable properties. Examplesof such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-beta;platelet-growth factor; transforming growth factors (TGFs) such asTGF-alpha and TGF-beta; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-alpha, beta, and -gamma; colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1 alpha,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12;IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; C5a; andother polypeptide factors including LIF and kit ligand (KL). As usedherein, the term cytokine includes proteins from natural sources or fromrecombinant cell culture, and biologically active equivalents of thenative sequence cytokines.

In an alternate embodiment, the Fc polypeptides of the present inventionare fused, conjugated, or operably linked to a toxin, including but notlimited to small molecule toxins and enzymatically active toxins ofbacterial, fungal, plant or animal origin, including fragments and/orvariants thereof. For example, a variety of immunotoxins and immunotoxinmethods are described in Thrush et al., 1996, Ann. Rev. Immunol.14:49-71. Small molecule toxins include but are not limited tocalicheamicin, maytansine (U.S. Pat. No. 5,208,020), trichothene, andCC1065. In one embodiment of the invention, the Fc polypeptide isconjugated to one or more maytansine molecules (e.g. about 1 to about 10maytansine molecules per antibody molecule). Maytansine may, forexample, be converted to May-SS-Me which may be reduced to May-SH3 andreacted with modified Fc polypeptide (Chari et al., 1992, CancerResearch 52: 127-131) to generate a maytansinoid-antibody ormaytansinoid-Fc fusion conjugate. Another conjugate of interestcomprises an Fc polypeptide, for example an antibody or Fc fusion,conjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. Structural analogues ofcalicheamicin that may be used include but are not limited to γ₁ ¹, α₂¹, α₃, N-acetyl-γ₁ ¹, PSAG, and Θ¹ ₁, (Hinman et al., 1993, CancerResearch 53:3336-3342; Lode et al., 1998, Cancer Research 58:2925-2928)(U.S. Pat. No. 5,714,586; U.S. Pat. No. 5,712,374; U.S. Pat. No.5,264,586; U.S. Pat. No. 5,773,001). Dolastatin 10 analogs such asauristatin E (AE) and monomethylauristatin E (MMAE) may find use asconjugates for the Fc variants of the present invention (Doronina etal., 2003, Nat Biotechnol 21(7):778-84; Francisco et al., 2003 Blood102(4):1458-65). Useful enyzmatically active toxins include but are notlimited to diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, PCT WO 93/21232,hereby incorporated by reference. The present invention furthercontemplates a conjugate between an Fc variant of the present inventionand a compound with nucleolytic activity, for example a ribonuclease orDNA endonuclease such as a deoxyribonuclease (Dnase).

In an alternate embodiment, an Fc variant of the present invention maybe fused, conjugated, or operably linked to a radioisotope to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugate antibodies and Fc fusions. Examplesinclude, but are not limited to, At211, 1131, 1125, Y90, Re186, Re188,Sm153, Bi212, P32, and radioactive isotopes of Lu. See for example,reference.

In yet another embodiment, an Fc variant of the present invention may beconjugated to a “receptor” (e.g., streptavidin) for utilization in tumorpretargeting wherein the Fc variant-receptor conjugate is administeredto the patient, followed by removal of unbound conjugate from thecirculation using a clearing agent and then administration of a “ligand”(e.g. avidin) which is conjugated to a cytotoxic agent (e.g. aradionucleotide). In an alternate embodiment, the Fc variant isconjugated or operably linked to an enzyme in order to employ AntibodyDependent Enzyme Mediated Prodrug Therapy (ADEPT). ADEPT may be used byconjugating or operably linking the Fc variant to a prodrug-activatingenzyme that converts a prodrug (e.g. a peptidyl chemotherapeutic agent,see PCT WO 81/01145) to an active anti-cancer drug. See, for example,PCT WO 88/07378 and U.S. Pat. No. 4,975,278. The enzyme component of theimmunoconjugate useful for ADEPT includes any enzyme capable of actingon a prodrug in such a way so as to covert it into its more active,cytotoxic form. Enzymes that are useful in the method of this inventioninclude but are not limited to alkaline phosphatase useful forconverting phosphate-containing prodrugs into free drugs; arylsulfataseuseful for converting sulfate-containing prodrugs into free drugs;cytosine deaminase useful for converting non-toxic 5-fluorocytosine intothe anti-cancer drug, 5-fluorouracil; proteases, such as serratiaprotease, thermolysin, subtilisin, carboxypeptidases and cathepsins(such as cathepsins B and L), that are useful for convertingpeptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases,useful for converting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as .beta.-galactosidase andneuramimidase useful for converting glycosylated prodrugs into freedrugs; beta-lactamase useful for converting drugs derivatized with.alpha.-lactams into free drugs; and penicillin amidases, such aspenicillin V amidase or penicillin G amidase, useful for convertingdrugs derivatized at their amine nitrogens with phenoxyacetyl orphenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs of the invention into free activedrugs (see, for example, Massey, 1987, Nature 328: 457-458). Fcvariant-abzyme conjugates can be prepared for delivery of the abzyme toa tumor cell population. A variety of additional conjugates arecontemplated for the Fc variants of the present invention. A variety ofchemotherapeutic agents, anti-angiogenic agents, tyrosine kinaseinhibitors, and other therapeutic agents are described below, which mayfind use as Fc variant conjugates.

Also contemplated as fusion and conjugate partners are Fc polypeptides.Thus an Fc variant may be a multimeric Fc polypeptide, comprising two ormore Fc regions. The advantage of such a molecule is that it providesmultiple binding sites for Fc receptors with a single protein molecule.In one embodiment, Fc regions may be linked using a chemical engineeringapproach. For example, Fab's and Fc's may be linked by thioether bondsoriginating at cysteine residues in the hinges, generating moleculessuch as FabFc₂ (Kan et al., 2001, J. Immunol., 2001, 166: 1320-1326;Stevenson et al., 2002, Recent Results Cancer Res. 159: 104-12; U.S.Pat. No. 5,681,566). Fc regions may be linked using disulfideengineering and/or chemical cross-linking, for example as described inCaron et al., 1992, J. Exp. Med. 176:1191-1195, and Shopes, 1992, J.Immunol. 148(9):2918-22. In a preferred embodiment, Fc regions may belinked genetically. For example multiple Cγ2 domains have been fusedbetween the Fab and Fc regions of an antibody (White et al., 2001,Protein Expression and Purification 21: 446-455). In a preferredembodiment, Fc regions in an Fc variant are linked genetically togenerated tandemly linked Fc regions as described in U.S. Ser. No.60/531,752, filed Dec. 22, 2003, entitled “Fc polypeptides with novel Fcreceptor binding sites”. Tandemly linked Fc polypeptides may comprisetwo or more Fc regions, preferably one to three, most preferably two Fcregions. It may be advantageous to explore a number of engineeringconstructs in order to obtain homo- or hetero-tandemly linked Fcvariants with the most favorable structural and functional properties.Tandemly linked Fc variants may be homo-tandemly linked Fc variants,that is an Fc variant of one isotype is fused genetically to another Fcvariant of the same isotype. It is anticipated that because there aremultiple FcγR, C1q, and/or FcRn binding sites on tandemly linked Fcpolypeptides, effector functions and/or pharmacokinetics may beenhanced. In an alternate embodiment, Fc variants from differentisotypes may be tandemly linked, referred to as hetero-tandemly linkedFc variants. For example, because of the capacity to target FcγR andFcαRI receptors, an Fc variant that binds both FcγRs and FcαRI mayprovide a significant clinical improvement.

As will be appreciated by one skilled in the art, in reality theconcepts and definitions of fusion and conjugate are overlapping. Thedesignation of an Fc variant as a fusion or conjugate is not meant toconstrain it to any particular embodiment of the present invention.Rather, these terms are used loosely to convey the broad concept thatany Fc variant of the present invention may be linked genetically,chemically, or otherwise, to one or more polypeptides or molecules toprovide some desirable property.

Fusion and conjugate partners may be linked to any region of an Fcvariant of the present invention, including at the N- or C-termini, orat some residue in-between the termini. In a preferred embodiment, afusion or conjugate partner is linked at the N- or C-terminus of the Fcvariant, most preferably the N-terminus. A variety of linkers may finduse in the present invention to covalently link Fc variants to a fusionor conjugate partner or generate an Fc fusion. By “linker”, “linkersequence”, “spacer”, “tethering sequence” or grammatical equivalentsthereof, herein is meant a molecule or group of molecules (such as amonomer or polymer) that connects two molecules and often serves toplace the two molecules in a preferred configuration. A number ofstrategies may be used to covalently link molecules together. Theseinclude, but are not limited to polypeptide linkages between N- andC-termini of proteins or protein domains, linkage via disulfide bonds,and linkage via chemical cross-linking reagents. In one aspect of thisembodiment, the linker is a peptide bond, generated by recombinanttechniques or peptide synthesis. Choosing a suitable linker for aspecific case where two polypeptide chains are to be connected dependson various parameters, including but not limited to the nature of thetwo polypeptide chains (e.g., whether they naturally oligomerize), thedistance between the N- and the C-termini to be connected if known,and/or the stability of the linker towards proteolysis and oxidation.Furthermore, the linker may contain amino acid residues that provideflexibility. Thus, the linker peptide may predominantly include thefollowing amino acid residues: Gly, Ser, Ala, or Thr. The linker peptideshould have a length that is adequate to link two molecules in such away that they assume the correct conformation relative to one another sothat they retain the desired activity. Suitable lengths for this purposeinclude at least one and not more than 50 amino acid residues.Preferably, the linker is from about 1 to 30 amino acids in length, withlinkers of 1 to 20 amino acids in length being most preferred. Inaddition, the amino acid residues selected for inclusion in the linkerpeptide should exhibit properties that do not interfere significantlywith the activity of the polypeptide. Thus, the linker peptide on thewhole should not exhibit a charge that would be inconsistent with theactivity of the polypeptide, or interfere with internal folding, or formbonds or other interactions with amino acid residues in one or more ofthe monomers that would seriously impede the binding of receptor monomerdomains. Useful linkers include glycine-serine polymers (including, forexample, (GS)n, (GSGGS)n SEQ ID NO:9, (GGGGS)n SEQ ID NO:10, and (GGGS)nSEQ ID NO:11, where n is an integer of at least one), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers such asthe tether for the shaker potassium channel, and a large variety ofother flexible linkers, as will be appreciated by those in the art.Glycine-serine polymers are preferred since both of these amino acidsare relatively unstructured, and therefore may be able to serve as aneutral tether between components. Secondly, serine is hydrophilic andtherefore able to solubilize what could be a globular glycine chain.Third, similar chains have been shown to be effective in joiningsubunits of recombinant proteins such as single chain antibodies.Suitable linkers may also be identified by screening databases of knownthree-dimensional structures for naturally occurring motifs that canbridge the gap between two polypeptide chains. In a preferredembodiment, the linker is not immunogenic when administered in a humanpatient. Thus linkers may be chosen such that they have lowimmunogenicity or are thought to have low immunogenicity. For example, alinker may be chosen that exists naturally in a human. In a mostpreferred embodiment, the linker has the sequence of the hinge region ofan antibody, that is the sequence that links the antibody Fab and Fcregions; alternatively the linker has a sequence that comprises part ofthe hinge region, or a sequence that is substantially similar to thehinge region of an antibody. Another way of obtaining a suitable linkeris by optimizing a simple linker, e.g., (Gly4Ser)n SEQ ID NO:10, throughrandom mutagenesis. Alternatively, once a suitable polypeptide linker isdefined, additional linker polypeptides can be created to select aminoacids that more optimally interact with the domains being linked. Othertypes of linkers that may be used in the present invention includeartificial polypeptide linkers and inteins. In another embodiment,disulfide bonds are designed to link the two molecules. In anotherembodiment, linkers are chemical cross-linking agents. For example, avariety of bifunctional protein coupling agents may be used, includingbut not limited to N-succinimidyl-3-(2-pyridyldithiol) propionate(SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., 1971, Science 238:1098.Chemical linkers may enable chelation of an isotope. For example,Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody (see PCT WO 94/11026).The linker may be cleavable, facilitating release of the cytotoxic drugin the cell. For example, an acid-labile linker, peptidase-sensitivelinker, dimethyl linker or disulfide-containing linker (Chari et al.,1992, Cancer Research 52: 127-131) may be used. Alternatively, a varietyof nonproteinaceous polymers, including but not limited to polyethyleneglycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers ofpolyethylene glycol and polypropylene glycol, may find use as linkers,that is may find use to link the Fc variants of the present invention toa fusion or conjugate partner to generate an Fc fusion, or to link theFc variants of the present invention to a conjugate.

Engineering Methods

Design strategies, computational screening methods, and librarygeneration methods are described in U.S. Ser. No. 10/672,280 and U.S.Ser. No. 10/822,231, entitled “Optimized Fc Variants and Methods fortheir Generation”, herein expressly incorporated by reference. Thesestrategies, approaches, techniques, and methods may be appliedindividually or in various combinations to generate optimized Fcvariants.

Experimental Production of Fc Variants

The present invention provides methods for producing and experimentallytesting Fc variants. The described methods are not meant to constrainthe present invention to any particular application or theory ofoperation. Rather, the provided methods are meant to illustrategenerally that one or more Fc variants may be produced andexperimentally tested to obtain variant Fc variants. General methods forantibody molecular biology, expression, purification, and screening aredescribed in Antibody Engineering, edited by Duebel & Kontermann,Springer-Verlag, Heidelberg, 2001; and Hayhurst & Georgiou, 2001, CurrOpin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng2:339-76; Antibodies: A Laboratory Manual by Harlow & Lane, New York:Cold Spring Harbor Laboratory Press, 1988.

In one embodiment of the present invention, nucleic acids are createdthat encode the Fc variants, and that may then be cloned into hostcells, expressed and assayed, if desired. Thus, nucleic acids, andparticularly DNA, may be made that encode each protein sequence. Thesepractices are carried out using well-known procedures. For example, avariety of methods that may find use in the present invention aredescribed in Molecular Cloning—A Laboratory Manual, 3^(rd) Ed.(Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001), andCurrent Protocols in Molecular Biology (John Wiley & Sons). As will beappreciated by those skilled in the art, the generation of exactsequences for a library comprising a large number of sequences ispotentially expensive and time consuming. Accordingly, there are avariety of techniques that may be used to efficiently generate librariesof the present invention. Such methods that may find use in the presentinvention are described or referenced in U.S. Pat. No. 6,403,312; U.S.Ser. No. 09/782,004; U.S. Ser. No. 09/927,790; U.S. Ser. No. 10/218,102;PCT WO 01/40091; and PCT WO 02/25588. Such methods include but are notlimited to gene assembly methods, PCR-based method and methods which usevariations of PCR, ligase chain reaction-based methods, pooled oligomethods such as those used in synthetic shuffling, error-proneamplification methods and methods which use oligos with randommutations, classical site-directed mutagenesis methods, cassettemutagenesis, and other amplification and gene synthesis methods. As isknown in the art, there are a variety of commercially available kits andmethods for gene assembly, mutagenesis, vector subcloning, and the like,and such commercial products find use in the present invention forgenerating nucleic acids that encode Fc variants.

The Fc variants of the present invention may be produced by culturing ahost cell transformed with nucleic acid, preferably an expressionvector, containing nucleic acid encoding the Fc variants, under theappropriate conditions to induce or cause expression of the protein. Theconditions appropriate for expression will vary with the choice of theexpression vector and the host cell, and will be easily ascertained byone skilled in the art through routine experimentation. A wide varietyof appropriate host cells may be used, including but not limited tomammalian cells, bacteria, insect cells, and yeast. For example, avariety of cell lines that may find use in the present invention aredescribed in the ATCC® cell line catalog, available from the AmericanType Culture Collection.

In a preferred embodiment, the Fc variants are expressed in mammalianexpression systems, including systems in which the expression constructsare introduced into the mammalian cells using virus such as retrovirusor adenovirus. Any mammalian cells may be used, with human, mouse, rat,hamster, and primate cells being particularly preferred. Suitable cellsalso include known research cells, including but not limited to Jurkat Tcells, NIH3T3, CHO, BHK, COS, HEK293, PER C.6, HeLa, Sp2/0, NS0 cellsand variants thereof. In an alternately preferred embodiment, libraryproteins are expressed in bacterial cells. Bacterial expression systemsare well known in the art, and include Escherichia coli (E. coli),Bacillus subtilis, Streptococcus cremoris, and Streptococcus lividans.In alternate embodiments, Fc variants are produced in insect cells (e.g.Sf21/Sf9, Trichoplusia ni Bti-Tn5b1-4) or yeast cells (e.g. S.cerevisiae, Pichia, etc). In an alternate embodiment, Fc variants areexpressed in vitro using cell free translation systems. In vitrotranslation systems derived from both prokaryotic (e.g. E. coli) andeukaryotic (e.g. wheat germ, rabbit reticulocytes) cells are availableand may be chosen based on the expression levels and functionalproperties of the protein of interest. For example, as appreciated bythose skilled in the art, in vitro translation is required for somedisplay technologies, for example ribosome display. In addition, the Fcvariants may be produced by chemical synthesis methods. Also transgenicexpression systems both animal (e.g. cow, sheep or goat milk,embryonated hen's eggs, whole insect larvae, etc.) and plant (e.g. corn,tobacco, duckweed, etc.)

The nucleic acids that encode the Fc variants of the present inventionmay be incorporated into an expression vector in order to express theprotein. A variety of expression vectors may be utilized for proteinexpression. Expression vectors may comprise self-replicatingextra-chromosomal vectors or vectors which integrate into a host genome.Expression vectors are constructed to be compatible with the host celltype. Thus expression vectors which find use in the present inventioninclude but are not limited to those which enable protein expression inmammalian cells, bacteria, insect cells, yeast, and in in vitro systems.As is known in the art, a variety of expression vectors are available,commercially or otherwise, that may find use in the present inventionfor expressing Fc variants.

Expression vectors typically comprise a protein operably linked withcontrol or regulatory sequences, selectable markers, any fusionpartners, and/or additional elements. By “operably linked” herein ismeant that the nucleic acid is placed into a functional relationshipwith another nucleic acid sequence. Generally, these expression vectorsinclude transcriptional and translational regulatory nucleic acidoperably linked to the nucleic acid encoding the Fc variant, and aretypically appropriate to the host cell used to express the protein. Ingeneral, the transcriptional and translational regulatory sequences mayinclude promoter sequences, ribosomal binding sites, transcriptionalstart and stop sequences, translational start and stop sequences, andenhancer or activator sequences. As is also known in the art, expressionvectors typically contain a selection gene or marker to allow theselection of transformed host cells containing the expression vector.Selection genes are well known in the art and will vary with the hostcell used.

Fc variants may be operably linked to a fusion partner to enabletargeting of the expressed protein, purification, screening, display,and the like. Fusion partners may be linked to the Fc variant sequencevia a linker sequences. The linker sequence will generally comprise asmall number of amino acids, typically less than ten, although longerlinkers may also be used. Typically, linker sequences are selected to beflexible and resistant to degradation. As will be appreciated by thoseskilled in the art, any of a wide variety of sequences may be used aslinkers. For example, a common linker sequence comprises the amino acidsequence GGGGS SEQ ID NO. 10. A fusion partner may be a targeting orsignal sequence that directs Fc variant and any associated fusionpartners to a desired cellular location or to the extracellular media.As is known in the art, certain signaling sequences may target a proteinto be either secreted into the growth media, or into the periplasmicspace, located between the inner and outer membrane of the cell. Afusion partner may also be a sequence that encodes a peptide or proteinthat enables purification and/or screening. Such fusion partners includebut are not limited to polyhistidine tags (His-tags) (for example H₆ andH₁₀ or other tags for use with Immobilized Metal Affinity Chromatography(IMAC) systems (e.g. Ni⁺² affinity columns)), GST fusions, MBP fusions,Strep-tag, the BSP biotinylation target sequence of the bacterial enzymeBirA, and epitope tags which are targeted by antibodies (for examplec-myc tags, flag-tags, and the like). As will be appreciated by thoseskilled in the art, such tags may be useful for purification, forscreening, or both. For example, an Fc variant may be purified using aHis-tag by immobilizing it to a Ni⁺² affinity column, and then afterpurification the same His-tag may be used to immobilize the antibody toa Ni⁺² coated plate to perform an ELISA or other binding assay (asdescribed below). A fusion partner may enable the use of a selectionmethod to screen Fc variants (see below). Fusion partners that enable avariety of selection methods are well-known in the art, and all of thesefind use in the present invention. For example, by fusing the members ofan Fc variant library to the gene III protein, phage display can beemployed (Kay et al., Phage display of peptides and proteins: alaboratory manual, Academic Press, San Diego, Calif., 1996; Lowman etal., 1991, Biochemistry 30:10832-10838; Smith, 1985, Science228:1315-1317). Fusion partners may enable Fc variants to be labeled.Alternatively, a fusion partner may bind to a specific sequence on theexpression vector, enabling the fusion partner and associated Fc variantto be linked covalently or noncovalently with the nucleic acid thatencodes them. For example, U.S. Ser. No. 09/642,574; U.S. Ser. No.10/080,376; U.S. Ser. No. 09/792,630; U.S. Ser. No. 10/023,208; U.S.Ser. No. 09/792,626; U.S. Ser. No. 10/082,671; U.S. Ser. No. 09/953,351;U.S. Ser. No. 10/097,100; U.S. Ser. No. 60/366,658; PCT WO 00/22906; PCTWO 01/49058; PCT WO 02/04852; PCT WO 02/04853; PCT WO 02/08023; PCT WO01/28702; and PCT WO 02/07466 describe such a fusion partner andtechnique that may find use in the present invention.

The methods of introducing exogenous nucleic acid into host cells arewell known in the art, and will vary with the host cell used. Techniquesinclude but are not limited to dextran-mediated transfection, calciumphosphate precipitation, calcium chloride treatment, polybrene mediatedtransfection, protoplast fusion, electroporation, viral or phageinfection, encapsulation of the polynucleotide(s) in liposomes, anddirect microinjection of the DNA into nuclei. In the case of mammaliancells, transfection may be either transient or stable.

In a preferred embodiment, Fc variants are purified or isolated afterexpression. Proteins may be isolated or purified in a variety of waysknown to those skilled in the art. Standard purification methods includechromatographic techniques, including ion exchange, hydrophobicinteraction, affinity, sizing or gel filtration, and reversed-phase,carried out at atmospheric pressure or at high pressure using systemssuch as FPLC and HPLC. Purification methods also includeelectrophoretic, immunological, precipitation, dialysis, andchromatofocusing techniques. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.As is well known in the art, a variety of natural proteins bind Fc andantibodies, and these proteins can find use in the present invention forpurification of Fc variants. For example, the bacterial proteins A and Gbind to the Fc region. Likewise, the bacterial protein L binds to theFab region of some antibodies, as of course does the antibody's targetantigen. Purification can often be enabled by a particular fusionpartner. For example, Fc variants may be purified using glutathioneresin if a GST fusion is employed, Ni⁺² affinity chromatography if aHis-tag is employed, or immobilized anti-flag antibody if a flag-tag isused. For general guidance in suitable purification techniques, seeProtein Purification Principles and Practice, 3^(rd) Ed., Scopes,Springer-Verlag, NY, 1994. The degree of purification necessary willvary depending on the screen or use of the Fc variants. In someinstances no purification is necessary. For example in one embodiment,if the Fc variants are secreted, screening may take place directly fromthe media. As is well known in the art, some methods of selection do notinvolve purification of proteins. Thus, for example, if a library of Fcvariants is made into a phage display library, protein purification maynot be performed.

Experimental Assays

Fc variants may be screened using a variety of methods, including butnot limited to those that use in vitro assays, in vivo and cell-basedassays, and selection technologies. Automation and high-throughputscreening technologies may be utilized in the screening procedures.Screening may employ the use of a fusion partner or label. The use offusion partners has been discussed above. By “labeled” herein is meantthat the Fc variants of the invention have one or more elements,isotopes, or chemical compounds attached to enable the detection in ascreen. In general, labels fall into three classes: a) immune labels,which may be an epitope incorporated as a fusion partner that isrecognized by an antibody, b) isotopic labels, which may be radioactiveor heavy isotopes, and c) small molecule labels, which may includefluorescent and colorimetric dyes, or molecules such as biotin thatenable other labeling methods. Labels may be incorporated into thecompound at any position and may be incorporated in vitro or in vivoduring protein expression.

In a preferred embodiment, the functional and/or biophysical propertiesof Fc variants are screened in an in vitro assay. In vitro assays mayallow a broad dynamic range for screening properties of interest.Properties of Fc variants that may be screened include but are notlimited to stability, solubility, and affinity for Fc ligands, forexample FcγRs. Multiple properties may be screened simultaneously orindividually. Proteins may be purified or unpurified, depending on therequirements of the assay. In one embodiment, the screen is aqualitative or quantitative binding assay for binding of Fc variants toa protein or nonprotein molecule that is known or thought to bind the Fcvariant. In a preferred embodiment, the screen is a binding assay formeasuring binding to the Target antigen. In an alternately preferredembodiment, the screen is an assay for binding of Fc variants to an Fcligand, including but are not limited to the family of FcγRs, theneonatal receptor FcRn, the complement protein C1q, and the bacterialproteins A and G. Said Fc ligands may be from any organism, with humans,mice, rats, rabbits, and monkeys preferred. Binding assays can becarried out using a variety of methods known in the art, including butnot limited to FRET (Fluorescence Resonance Energy Transfer) and BRET(Bioluminescence Resonance Energy Transfer)-based assays, AlphaScreen™(Amplified Luminescent Proximity Homogeneous Assay), ScintillationProximity Assay, ELISA (Enzyme-Linked ImmunoSorbent Assay), SPR (SurfacePlasmon Resonance, also known as BIACORE®), isothermal titrationcalorimetry, differential scanning calorimetry, gel electrophoresis, andchromatography including gel filtration. These and other methods maytake advantage of some fusion partner or label of the Fc variant. Assaysmay employ a variety of detection methods including but not limited tochromogenic, fluorescent, luminescent, or isotopic labels.

The biophysical properties of Fc variants, for example stability andsolubility, may be screened using a variety of methods known in the art.Protein stability may be determined by measuring the thermodynamicequilibrium between folded and unfolded states. For example, Fc variantsof the present invention may be unfolded using chemical denaturant,heat, or pH, and this transition may be monitored using methodsincluding but not limited to circular dichroism spectroscopy,fluorescence spectroscopy, absorbance spectroscopy, NMR spectroscopy,calorimetry, and proteolysis. As will be appreciated by those skilled inthe art, the kinetic parameters of the folding and unfolding transitionsmay also be monitored using these and other techniques. The solubilityand overall structural integrity of an Fc variant may be quantitativelyor qualitatively determined using a wide range of methods that are knownin the art. Methods which may find use in the present invention forcharacterizing the biophysical properties of Fc variants include gelelectrophoresis, isoelectric focusing, capillary electrophoresis,chromatography such as size exclusion chromatography, ion-exchangechromatography, and reversed-phase high performance liquidchromatography, peptide mapping, oligosaccharide mapping, massspectrometry, ultraviolet absorbance spectroscopy, fluorescencespectroscopy, circular dichroism spectroscopy, isothermal titrationcalorimetry, differential scanning calorimetry, analyticalultra-centrifugation, dynamic light scattering, proteolysis, andcross-linking, turbidity measurement, filter retardation assays,immunological assays, fluorescent dye binding assays, protein-stainingassays, microscopy, and detection of aggregates via ELISA or otherbinding assay. Structural analysis employing X-ray crystallographictechniques and NMR spectroscopy may also find use. In one embodiment,stability and/or solubility may be measured by determining the amount ofprotein solution after some defined period of time. In this assay, theprotein may or may not be exposed to some extreme condition, for exampleelevated temperature, low pH, or the presence of denaturant. Becausefunction typically requires a stable, soluble, and/orwell-folded/structured protein, the aforementioned functional andbinding assays also provide ways to perform such a measurement. Forexample, a solution comprising an Fc variant could be assayed for itsability to bind target antigen, then exposed to elevated temperature forone or more defined periods of time, then assayed for antigen bindingagain. Because unfolded and aggregated protein is not expected to becapable of binding antigen, the amount of activity remaining provides ameasure of the Fc variant's stability and solubility.

In a preferred embodiment, the library is screened using one or morecell-based or in vitro assays. For such assays, Fc variants, purified orunpurified, are typically added exogenously such that cells are exposedto individual variants or groups of variants belonging to a library.These assays are typically, but not always, based on the biology of theability of the antibody or Fc fusion to bind to the target antigen andmediate some biochemical event, for example effector functions likecellular lysis, phagocytosis, ligand/receptor binding inhibition,inhibition of growth and/or proliferation, apoptosis and the like. Suchassays often involve monitoring the response of cells to Fc variant, forexample cell survival, cell death, cellular phagocytosis, cell lysis,change in cellular morphology, or transcriptional activation such ascellular expression of a natural gene or reporter gene. For example,such assays may measure the ability of Fc variants to elicit ADCC, ADCP,or CDC. For some assays additional cells or components, that is inaddition to the target cells, may need to be added, for example serumcomplement, or effector cells such as peripheral blood monocytes(PBMCs), NK cells, macrophages, and the like. Such additional cells maybe from any organism, preferably humans, mice, rat, rabbit, and monkey.Crosslinked or monomeric antibodies and Fc fusions may cause apoptosisof certain cell lines expressing the antibody's target antigen, or theymay mediate attack on target cells by immune cells which have been addedto the assay. Methods for monitoring cell death or viability are knownin the art, and include the use of dyes, fluorophores, immunochemical,cytochemical, and radioactive reagents. For example, caspase assays orannexin-flourconjugates may enable apoptosis to be measured, and uptakeor release of radioactive substrates (e.g. Chromium-51 release assays)or the metabolic reduction of fluorescent dyes such as alamar blue mayenable cell growth, proliferationor activation to be monitored. In apreferred embodiment, the DELFIA® EuTDA-based cytotoxicity assay (PerkinElmer, Mass.) is used. Alternatively, dead or damaged target cells maybe monitored by measuring the release of one or more naturalintracellular proteins, for example lactate dehydrogenase.Transcriptional activation may also serve as a method for assayingfunction in cell-based assays. In this case, response may be monitoredby assaying for natural genes or proteins which may be upregulated ordown-regulated, for example the release of certain interleukins may bemeasured, or alternatively readout may be via a luciferase orGFP-reporter construct. Cell-based assays may also involve the measureof morphological changes of cells as a response to the presence of an Fcvariant. Cell types for such assays may be prokaryotic or eukaryotic,and a variety of cell lines that are known in the art may be employed.Alternatively, cell-based screens are performed using cells that havebeen transformed or transfected with nucleic acids encoding the Fcvariants.

In vitro assays include but are not limited to binding assays, ADCC,CDC, cytotoxicity, proliferation, peroxide/ozone release, chemotaxis ofeffector cells, inhibition of such assays by reduced effector functionantibodies; ranges of activities such as >100× improvement or >100×reduction, blends of receptor activation and the assay outcomes that areexpected from such receptor profiles.

Pre-Clinical Experiments and Animal Models

The biological properties of the Fc variants of the present inventionmay be characterized in cell, tissue, and whole organism experiments. Asis know in the art, drugs are often tested in animals, including but notlimited to mice, rats, rabbits, dogs, cats, pigs, and monkeys, in orderto measure a drug's efficacy for treatment against a disease or diseasemodel, or to measure a drug's pharmacokinetics, toxicity, and otherproperties. Said animals may be referred to as disease models. Withrespect to the Fc variants of the present invention, a particularchallenge arises when using animal models to evaluate the potential forin-human efficacy of candidate polypeptides—this is due, at least inpart, to the fact that Fc variants that have a specific effect on theaffinity for a human Fc receptor may not have a similar affinity effectwith the orthologous animal receptor. These problems can be furtherexacerbated by the inevitable ambiguities associated with correctassignment of true orthologues (Mechetina et al., Immunogenetics, 200254:463-468), and the fact that some orthologues simply do not exist inthe animal (for example, humans possess an FcγRIIa whereas mice do not).Therapeutics are often tested in mice, including but not limited to nudemice, SCID mice, xenograft mice, and transgenic mice (including knockinsand knockouts). For example, an antibody or Fc fusion of the presentinvention that is intended as an anti-cancer therapeutic may be testedin a mouse cancer model, for example a xenograft mouse. In this method,a tumor or tumor cell line is grafted onto or injected into a mouse, andsubsequently the mouse is treated with the therapeutic to determine theability of the antibody or Fc fusion to reduce or inhibit cancer growthand metastasis. An alternative approach is the use of a SCID murinemodel in which immune-deficient mice are injected with human PBLs,conferring a semi-functional and human immune system—with an appropriatearray of human FcγRs—to the mice that have subsequently been injectedwith antibodies or Fc polypeptides that target injected human tumorcells. In such a model, the Fc polypeptides that target the desiredantigen (such as her2/neu on SkOV3 ovarian cancer cells) interact withhuman PBLs within the mice to engage tumoricidal effector functions.Such experimentation may provide meaningful data for determination ofthe potential of said Fc variant to be used as a therapeutic. Anyorganism, preferably mammals, may be used for testing. For examplebecause of their genetic similarity to humans, monkeys can be suitabletherapeutic models, and thus may be used to test the efficacy, toxicity,pharmacokinetics, or other property of the Fc polypeptides of thepresent invention. Tests of the Fc variants of the present invention inhumans are ultimately required for approval as drugs, and thus of coursethese experiments are contemplated. Thus the Fc variants of the presentinvention may be tested in humans to determine their therapeuticefficacy, toxicity, pharmacokinetics, and/or other clinical properties.

The Fc variants of the present invention may confer superior performanceon Fc polypeptides therapeutics in animal models or in humans. Thereceptor binding profiles of such Fc variants, as described in thisspecification, may, for example, be selected to increase the potency ofcytotoxic drugs or to target specific effector functions or effectorcells to improve the selectivity of the drug's action. Further, receptorbinding profiles can be selected that may reduce some or all effectorfunctions thereby reducing the side-effects or toxicity of such Fcpolypeptide drugs. For example, an Fc variant with reduced binding toFcγRIIIa, FcγRI and FcγRIIa can be selected to eliminate mostcell-mediated effector function, or an Fc variant with reduced bindingto C1q may be selected to limit complement-mediated effector functions.In some contexts, such effector functions are known to have potentialtoxic effects, therefore eliminating them may increase the safety of theFc polypeptide drug, and such improved safety may be characterized inanimal models. In some contexts, such effector functions are known tomediate the desirable therapeutic activity, therefore enhancing them mayincrease the activity or potency of the Fc polypeptide drug and suchimproved activity or potency may be characterized in animal models.

Optimized Fc variants can be tested in a variety of orthotopic tumormodels. These clinically relevant animal models are important in thestudy of pathophysiology and therapy of aggressive cancers likepancreatic, prostate and breast cancer. Immune deprived mice including,but not limited to athymic nude or SCID mice are frequently used inscoring of local and systemic tumor spread from the site of intraorgan(e.g. pancreas, prostate or mammary gland) injection of human tumorcells or fragments of donor patients.

In preferred embodiments, Fc variants of the present invention may beassessed for efficacy in clinically relevant animal models of varioushuman diseases. In many cases, relevant models include varioustransgenic animals for specific tumor antigens. Relevant transgenicmodels such as those that express human Fc receptors (e.g., FcγRIIIaincluding the gamma chain, FcγRI, FcγRIIa, FcγRIIb, and others) could beused to evaluate and test the efficacy of Fc polypeptides of the presentinvention. Evaluation of Fc variants by the introduction of human geneswhich directly or indirectly mediate effector function in mice or otherrodents, may enable physiological studies of efficacy in tumor toxicityor other diseases such as autoimmune disorders and RA. Human Fcreceptors such as FcγRIIIa may possess polymorphisms, such as that atposition 158 (V or F as described) which would further enable theintroduction of specific and combinations of human polymorphisms intorodents. The various studies involving polymorphism-specific FcγRs isnot limited to this section, however, and encompasses all discussionsand applications of FcγRs in general as specified in throughout thisapplication. Fc variants of the present invention may confer superioractivity on Fc polypeptides in such transgenic models. In particular,variants with binding profiles optimized for human FcγRIIIa mediatedactivity may show superior activity in transgenic CD16 (FcγRIII) mice.Similar improvements in efficacy in mice transgenic for the other humanFc receptors, e.g. FcγRIIa, FcγRI, etc., may be observed for Fc variantswith binding profiles optimized for the respective receptors. Micetransgenic for multiple human receptors would show improved activity forFc variants with binding profiles optimized for the correspondingmultiple receptors, for example as outlined in Table 1.

The introduction of target tumor antigens such as human CD20 into rodentB-cells in the form of a transgenic animal model can be used to providea more relevant evaluation of efficacy. As such, the target antigen neednot be limited to a fully human construct but could be a fusion proteincontaining the relevant human epitope of the target antigen. In apreferred embodiment, the testing of Fc polypeptides may includetransgenic model systems, which include the combination of but notlimited to both human target antigen and human Fc receptors (e.g. CD16and other related receptors mediating effector functions) to evaluateefficacy and tumoricidal activity.

In a preferred embodiment, Fc polypeptides of the present invention thattarget the Her2 antigen (e.g. Fc variants of mu4D5 or its humanizedanalogues) may be assessed for efficacy in a clinically relevant mousemodel of breast cancer. Examples of relevant models include, but are notlimited to: 1) the HER2/neu (neu-N)-transgenic mice, which are derivedfrom the parental FVB/N mouse strain and are transgenic for the rat formof the proto-oncogene HER2/neu (neu); and 2) transgenic mice thatoverexpress human HER2 under the murine mammary tumor virus promoter(Finkle et al., 2004, Clin Cancer Res. 10 (7):2499-511). Fc polypeptidesof the present invention that show superior efficacy in these modelsrepresent likely candidates for further development.

Because of the difficulties and ambiguities associated with using animalmodels to characterize the potential efficacy of candidate therapeuticantibodies in a human patient, some variant polypeptides of the presentinvention may find utility as proxies for assessing potential in-humanefficacy. Such proxy molecules would preferably mimic, in the animalsystem, the FcγR and/or complement biology of a corresponding candidatehuman Fc variant. This mimicry is most likely to be manifested byrelative association affinities between specific Fc variants and animalvs. human receptors. For example, if one were using a mouse model toassess the potential in-human efficacy of an Fc variant that hasenhanced affinity for human FcγRIIIa, an appropriate proxy variant wouldhave enhanced affinity for mouse FcγRIII-2 (mouse CD16-2). Alternativelyif one were using a mouse model to assess the potential in-humanefficacy of an Fc variant that has reduced affinity for the humaninhibitory receptor FcγRIIb, an appropriate proxy variant would havereduced affinity for mouse FcγRII. It should also be noted that theproxy Fc variants could be created in the context of a human Fc variant,an animal Fc variant, or both.

In a preferred embodiment, the testing of Fc variants may include studyof efficacy in primates (e.g. cynomolgus monkey model) to facilitate theevaluation of depletion of specific target cells harboring targetantigen. Additional primate models include but not limited to that ofthe rhesus monkey and Fc polypeptides in therapeutic studies ofautoimmune, transplantation and cancer.

Toxicity studies are performed to determine the Fc polypeptide relatedeffects that cannot be evaluated in standard pharmacology profile oroccur only after repeated administration of the agent. Most toxicitytests are performed in two species—a rodent and a non-rodent—to ensurethat any unexpected adverse effects are not overlooked before newtherapeutic entities are introduced into humans. In general, thesemodels may measure a variety of toxicities including genotoxicity,chronic toxicity, immunogenicity, reproductive/developmental toxicityand carcinogenicity. Included within the aforementioned parameters arestandard measurement of food consumption, bodyweight, antibodyformation, clinical chemistry, and macro- and microscopic examination ofstandard organs/tissues (e.g. cardiotoxicity). Additional parameters ofmeasurement are injection site trauma and the measurement ofneutralizing antibodies, if any. Traditionally, monoclonal antibodytherapeutics, naked or conjugated are evaluated for cross-reactivitywith normal tissues, immunogenicity/antibody production, conjugate orlinker toxicity and “bystander” toxicity of radiolabeled species.Nonetheless, such studies may have to be individualized to addressspecific concerns and following the guidance set by ICH S6 (Safetystudies for biotechnological products also noted above). As such, thegeneral principles are that the products are sufficiently wellcharacterized and for which impurities/contaminants have been removed,that the test material is comparable throughout development, and GLPcompliance.

The pharmacokinetics (PK) of the Fc variants of the invention can bestudied in a variety of animal systems, with the most relevant beingnon-human primates such as the cynomolgus, rhesus monkeys. Single orrepeated i.v./s.c. administrations over a dose range of 6000-fold(0.05-300 mg/kg) can be evaluated for the half-life (days to weeks)using plasma concentration and clearance as well as volume ofdistribution at a steady state and level of systemic absorbance can bemeasured. Examples of such parameters of measurement generally includemaximum observed plasma concentration (Cmax), the time to reach Cmax(Tmax), the area under the plasma concentration-time curve from time 0to infinity [AUC(0-inf] and apparent elimination half-life (T½).Additional measured parameters could include compartmental analysis ofconcentration-time data obtained following i.v. administration andbioavailability. Examples of pharmacological/toxicological studies usingcynomolgus have been established for Rituxan and Zevalin in whichmonoclonal antibodies to CD20 are cross-reactive. Biodistribution,dosimetry (for radiolabeled antibodies or Fc fusions), and PK studiescan also be done in rodent models. Such studies would evaluate toleranceat all doses administered, toxicity to local tissues, preferentiallocalization to rodent xenograft animal models, depletion of targetcells (e.g. CD20 positive cells).

The Fc variants of the present invention may confer superiorpharmacokinetics on Fc polypeptide therapeutics in animal systems or inhumans. For example, increased binding to FcRn may increase thehalf-life and exposure of the Fc polypeptide. Alternatively, decreasedbinding to FcRn may decrease the half-life and exposure of the Fcpolypeptide in cases where reduced exposure is favorable, such as whensuch drug has side-effects.

It is known in the art that the array of Fc receptors is differentiallyexpressed on various immune cell types, as well as in different tissues.Differential tissue distribution of Fc receptors may ultimately have animpact on the pharmacodynamic (PD) and pharmacokinetic (PK) propertiesof Fc variants of the present invention. Because Fc variants of thepresentation have varying affinities for the array of Fc receptors,further screening of the polypeptides for PD and/or PK properties may beextremely useful for defining the optimal balance of PD, PK, andtherapeutic efficacy conferred by each candidate polypeptide.

Pharmacodynamic studies may include, but are not limited to, targetingspecific tumor cells or blocking signaling mechanisms, measuringdepletion of target antigen expressing cells or signals, etc. The Fcvariants of the present invention may target particular effector cellpopulations and thereby direct Fc polypeptides to recruit certainactivities to improve potency or to increase penetration into aparticularly favorable physiological compartment. For example,neutrophil activity and localization can be targeted by an Fc variantthat preferentially targets FcγRIIIb. Such pharmacodynamic effects maybe demonstrated in animal models or in humans.

Therapeutic Use of Fc Variants

The Fc variants of the present invention may be used for varioustherapeutic purposes. As will be appreciated by those in the art, the Fcvariants of the present invention may be used for any therapeuticpurpose for which antibodies, Fc fusions, and the like may be used. In apreferred embodiment, the Fc variants are administered to a patient totreat disorders including but not limited to autoimmune and inflammatorydiseases, infectious diseases, and cancer.

A “patient” for the purposes of the present invention includes bothhumans and other animals, preferably mammals and most preferably humans.Thus the Fc variants of the present invention have both human therapyand veterinary applications. The term “treatment” in the presentinvention is meant to include therapeutic treatment, as well asprophylactic, or suppressive measures for a disease or disorder. Thus,for example, successful administration of an Fc variant prior to onsetof the disease results in treatment of the disease. As another example,successful administration of an optimized Fc variant after clinicalmanifestation of the disease to combat the symptoms of the diseasecomprises treatment of the disease. “Treatment” also encompassesadministration of an optimized Fc variant after the appearance of thedisease in order to eradicate the disease. Successful administration ofan agent after onset and after clinical symptoms have developed, withpossible abatement of clinical symptoms and perhaps amelioration of thedisease, comprises treatment of the disease. Those “in need oftreatment” include mammals already having the disease or disorder, aswell as those prone to having the disease or disorder, including thosein which the disease or disorder is to be prevented.

In one embodiment, an Fc variant of the present invention isadministered to a patient having a disease involving inappropriateexpression of a protein or other molecule. Within the scope of thepresent invention this is meant to include diseases and disorderscharacterized by aberrant proteins, due for example to alterations inthe amount of a protein present, protein localization, posttranslationalmodification, conformational state, the presence of a mutant or pathogenprotein, etc. Similarly, the disease or disorder may be characterized byalterations molecules including but not limited to polysaccharides andgangliosides. An overabundance may be due to any cause, including butnot limited to overexpression at the molecular level, prolonged oraccumulated appearance at the site of action, or increased activity of aprotein relative to normal. Included within this definition are diseasesand disorders characterized by a reduction of a protein. This reductionmay be due to any cause, including but not limited to reduced expressionat the molecular level, shortened or reduced appearance at the site ofaction, mutant forms of a protein, or decreased activity of a proteinrelative to normal. Such an overabundance or reduction of a protein canbe measured relative to normal expression, appearance, or activity of aprotein, and said measurement may play an important role in thedevelopment and/or clinical testing of the Fc variants of the presentinvention.

“Cancer” and “cancerous” herein refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. Examples of cancer include but are not limited to carcinoma,lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrinetumors, mesothelioma, schwanoma, meningioma, adenocarcinoma, melanoma,and leukemia or lymphoid malignancies.

More particular examples of such cancers include hematologicmalignancies, such as Hodgkin's lymphoma; non-Hodgkin's lymphomas(Burkitt's lymphoma, small lymphocytic lymphoma/chronic lymphocyticleukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma,diffuse large B-cell lymphoma, marginal zone lymphoma, hairy cellleukemia and lymphoplamacytic leukemia), tumors of lymphocyte precursorcells, including B-cell acute lymphoblastic leukemia/lymphoma, andT-cell acute lymphoblastic leukemia/lymphoma, thymoma, tumors of themature T and NK cells, including peripheral T-cell leukemias, adultT-cell leukemia/T-cell lymphomas and large granular lymphocyticleukemia, Langerhans cell histocytosis, myeloid neoplasias such as acutemyelogenous leukemias, including AML with maturation, AML withoutdifferentiation, acute promyelocytic leukemia, acute myelomonocyticleukemia, and acute monocytic leukemias, myelodysplastic syndromes, andchronic myeloproliferative disorders, including chronic myelogenousleukemia; tumors of the central nervous system such as glioma,glioblastoma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma,and retinoblastoma; solid tumors of the head and neck (eg.nasopharyngeal cancer, salivary gland carcinoma, and esophageal cancer),lung (eg. small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung),digestive system (eg. gastric or stomach cancer includinggastrointestinal cancer, cancer of the bile duct or biliary tract, coloncancer, rectal cancer, colorectal cancer, and anal carcinoma),reproductive system (eg. testicular, penile, or prostate cancer,uterine, vaginal, vulval, cervical, ovarian, and endometrial cancer),skin (eg. melanoma, basal cell carcinoma, squamous cell cancer, actinickeratosis), liver (eg. liver cancer, hepatic carcinoma, hepatocellularcancer, and hepatoma), bone (eg. osteoclastoma, and osteolytic bonecancers) additional tissues and organs (eg. pancreatic cancer, bladdercancer, kidney or renal cancer, thyroid cancer, breast cancer, cancer ofthe peritoneum, and Kaposi's sarcoma), and tumors of the vascular system(eg. angiosarcoma and hemangiopericytoma).

“Autoimmune diseases” herein include allogenic islet graft rejection,alopecia areata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, antineutrophil cytoplasmic autoantibodies(ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolyticanemia, autoimmune hepatitis, autoimmune myocarditis, autoimmuneneutropenia, autoimmune oophoritis and orchitis, autoimmunethrombocytopenia, autoimmune urticaria, Behcet's disease, bullouspemphigoid, cardiomyopathy, Castleman's syndrome, celiacspruce-dermatitis, chronic fatigue immune disfunction syndrome, chronicinflammatory demyelinating polyneuropathy, Churg-Strauss syndrome,cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn'sdisease, dermatomyositis, discoid lupus, essential mixedcryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis,glomerulonephritis, Grave's disease, Guillain-Barre, Goodpasture'ssyndrome, graft-versus-host disease (GVHD), Hashimoto's thyroiditis,hemophilia A, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, IgM polyneuropathies, immune mediatedthrombocytopenia, juvenile arthritis, Kawasaki's disease, lichenplantus, lupus erthematosis, Meniere's disease, mixed connective tissuedisease, multiple sclerosis, type 1 diabetes mellitus, myastheniagravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychrondritis, polyglandular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobinulinemia, primarybiliary cirrhosis, psoriasis, psoriatic arthritis, Reynauld'sphenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis,scleroderma, Sjorgen's syndrome, solid organ transplant rejection,stiff-man syndrome, systemic lupus erythematosus, takayasu arteritis,temporal arteristis/giant cell arteritis, thrombotic thrombocytopeniapurpura, ulcerative colitis, uveitis, vasculitides such as dermatitisherpetiformis vasculitis, vitiligo, and Wegner's granulomatosis.

“Inflammatory disorders” herein include acute respiratory distresssyndrome (ARDS), acute septic arthritis, allergic encephalomyelitis,allergic rhinitis, allergic vasculitis, allergy, asthma,atherosclerosis, chronic inflammation due to chronic bacterial or viralinfectionis, chronic obstructive pulmonary disease (COPD), coronaryartery disease, encephalitis, inflammatory bowel disease, inflammatoryosteolysis, inflammation associated with acute and delayedhypersensitivity reactions, inflammation associated with tumors,peripheral nerve injury or demyelinating diseases, inflammationassociated with tissue trauma such as burns and ischemia, inflammationdue to meningitis, multiple organ injury syndrome, pulmonary fibrosis,sepsis and septic shock, Stevens-Johnson syndrome, undifferentiatedarthropy, and undifferentiated spondyloarthropathy.

“Infectious diseases” herein include diseases caused by pathogens suchas viruses, bacteria, fungi, protozoa, and parasites. Infectiousdiseases may be caused by viruses including adenovirus, cytomegalovirus,dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C,herpes simplex type I, herpes simplex type II, human immunodeficiencyvirus, (HIV), human papilloma virus (HPV), influenza, measles, mumps,papova virus, polio, respiratory syncytial virus, rinderpest,rhinovirus, rotavirus, rubella, SARS virus, smallpox, viral meningitis,and the like. Infections diseases may also be caused by bacteriaincluding Bacillus antracis, Borrelia burgdorferi, Campylobacter jejuni,Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani,Diptheria, E. coli, Legionella, Helicobacter pylori, Mycobacteriumrickettsia, Mycoplasma nesisseria, Pertussis, Pseudomonas aeruginosa, S.pneumonia, Streptococcus, Staphylococcus, Vibria cholerae, Yersiniapestis, and the like. Infectious diseases may also be caused by fungisuch as Aspergillus fumigatus, Blastomyces dermatitidis, Candidaalbicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasmacapsulatum, Penicillium marneffei, and the like. Infectious diseases mayalso be caused by protozoa and parasites such as chlamydia, kokzidioa,leishmania, malaria, rickettsia, trypanosoma, and the like.

Furthermore, Fc variants of the present invention may be used to preventor treat additional conditions including but not limited to heartconditions such as congestive heart failure (CHF), myocarditis and otherconditions of the myocardium; skin conditions such as rosecea, acne, andeczema; bone and tooth conditions such as bone loss, osteoporosis,Paget's disease, Langerhans' cell histiocytosis, periodontal disease,disuse osteopenia, osteomalacia, monostotic fibrous dysplasia,polyostotic fibrous dysplasia, bone metastasis, bone pain management,humoral malignant hypercalcemia, periodontal reconstruction, spinal cordinjury, and bone fractures; metabolic conditions such as Gaucher'sdisease; endocrine conditions such as Cushing's syndrome; andneurological conditions.

Formulation, Administration, and Dosing

Pharmaceutical compositions are contemplated wherein an Fc variant ofthe present invention and one or more therapeutically active agents areformulated. Formulations of the Fc variants of the present invention areprepared for storage by mixing said Fc variant having the desired degreeof purity with optional pharmaceutically acceptable carriers, excipientsor stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed., 1980), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, acetate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; sweeteners and other flavoring agents;fillers such as microcrystalline cellulose, lactose, corn and otherstarches; binding agents; additives; coloring agents; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). In a preferred embodiment, the pharmaceuticalcomposition that comprises the Fc variant of the present invention maybe in a water-soluble form, such as being present as pharmaceuticallyacceptable salts, which is meant to include both acid and base additionsalts. “Pharmaceutically acceptable acid addition salt” refers to thosesalts that retain the biological effectiveness of the free bases andthat are not biologically or otherwise undesirable, formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and the like, and organic acids suchas acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. “Pharmaceutically acceptable base additionsalts” include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Particularly preferred are theammonium, potassium, sodium, calcium, and magnesium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine. The formulations to beused for in vivo administration are preferrably sterile. This is readilyaccomplished by filtration through sterile filtration membranes or othermethods.

The Fc variants disclosed herein may also be formulated asimmunoliposomes. A liposome is a small vesicle comprising various typesof lipids, phospholipids and/or surfactant that is useful for deliveryof a therapeutic agent to a mammal. Liposomes containing the Fc variantare prepared by methods known in the art, such as described in Epsteinet al., 1985, Proc Natl Acad Sci USA, 82:3688; Hwang et al., 1980, ProcNatl Acad Sci USA, 77:4030; U.S. Pat. No. 4,485,045; U.S. Pat. No.4,544,545; and PCT WO 97/38731. Liposomes with enhanced circulation timeare disclosed in U.S. Pat. No. 5,013,556. The components of the liposomeare commonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes. Particularly useful liposomes canbe generated by the reverse phase evaporation method with a lipidcomposition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter. A chemotherapeutic agent or other therapeuticallyactive agent is optionally contained within the liposome (Gabizon etal., 1989, J National Cancer Inst 81:1484).

The Fc variant and other therapeutically active agents may also beentrapped in microcapsules prepared by methods including but not limitedto coacervation techniques, interfacial polymerization (for exampleusing hydroxymethylcellulose or gelatin-microcapsules, orpoly-(methylmethacylate) microcapsules), colloidal drug delivery systems(for example, liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules), and macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed., 1980. Sustained-release preparations may be prepared. Suitableexamples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymer, which matrices are in the form ofshaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for examplepoly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gammaethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT® (whichare injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), poly-D-(−)-3-hydroxybutyric acid, andProLease® (commercially available from Alkermes), which is amicrosphere-based delivery system composed of the desired bioactivemolecule incorporated into a matrix of poly-DL-lactide-co-glycolide(PLG).

Administration of the pharmaceutical composition comprising an Fcvariant of the present invention, preferably in the form of a sterileaqueous solution, may be done in a variety of ways, including, but notlimited to orally, subcutaneously, intravenously, intranasally,intraotically, transdermally, topically (e.g., gels, salves, lotions,creams, etc.), intraperitoneally, intramuscularly, intrapulmonary,vaginally, parenterally, rectally, or intraocularly. In some instances,for example for the treatment of wounds, inflammation, etc., the Fcvariant may be directly applied as a solution or spray. As is known inthe art, the pharmaceutical composition may be formulated accordinglydepending upon the manner of introduction.

Subcutaneous administration may be preferable in some circumstancesbecause the patient may self-administer the pharmaceutical composition.Many protein therapeutics are not sufficiently potent to allow forformulation of a therapeutically effective dose in the maximumacceptable volume for subcutaneous administration. This problem may beaddressed in part by the use of protein formulations comprisingarginine-HCl, histidine, and polysorbate (see WO 04091658). Fcpolypeptides of the present invention may be more amenable tosubcutaneous administration due to, for example, increased potency,improved serum half-life, or enhanced solubility.

As is known in the art, protein therapeutics are often delivered by IVinfusion or bolus. The Fc variants of the present invention may also bedelivered using such methods. For example, administration may venous beby intravenous infusion with 0.9% sodium chloride as an infusionvehicle.

Pulmonary delivery may be accomplished using an inhaler or nebulizer anda formulation comprising an aerosolizing agent. For example, AERx®inhalable technology commercially available from Aradigm, or Inhance™pulmonary delivery system commercially available from NektarTherapeutics may be used. Fc variants of the present invention may bemore amenable to intrapulmonary delivery. FcRn is present in the lung,and may promote transport from the lung to the bloodstream (e.g.Syntonix WO 04004798, Bitonti et.al. (2004) Proc. Nat. Acad. Sci.101:9763-8). Accordingly, antibodies or Fc fusions that bind FcRn moreeffectively in the lung or that are released more efficiently in thebloodstream may have improved bioavailability following intrapulmonaryadministration. Fc variants of the present invention may also be moreamenable to intrapulmonary administration due to, for example, improvedsolubility or altered isoelectric point.

Furthermore, Fc polypeptides of the present invention may be moreamenable to oral delivery due to, for example, improved stability atgastric pH and increased resistance to proteolysis. Furthermore, FcRnappears to be expressed in the intestinal epithelia of adults (Dickinsonet al., 1999, J Clin Invest 104:903-11), so Fc polypeptides of thepresent invention, for example antibodies or Fc fusions, with improvedFcRn interaction profiles may show enhanced bioavailability followingoral administration. FcRn mediated transport of Fc variants may alsooccur at other mucus membranes such as those in the gastrointestinal,respiratory, and genital tracts (Yoshida et al., 2004, Immunity20:769-83).

In addition, any of a number of delivery systems are known in the artand may be used to administer the Fc variants of the present invention.Examples include, but are not limited to, encapsulation in liposomes,microparticles, microspheres (eg. PLA/PGA microspheres), and the like.Alternatively, an implant of a porous, non-porous, or gelatinousmaterial, including membranes or fibers, may be used. Sustained releasesystems may comprise a polymeric material or matrix such as polyesters,hydrogels, poly(vinylalcohol),polylactides, copolymers of L-glutamicacid and ethyl-L-gutamate, ethylene-vinyl acetate, lactic acid-glycolicacid copolymers such as the LUPRON DEPOT®, andpoly-D-(−)-3-hydroxybutyric acid. It is also possible to administer anucleic acid encoding the Fc variant of the current invention, forexample by retroviral infection, direct injection, or coating withlipids, cell surface receptors, or other transfection agents. In allcases, controlled release systems may be used to release the Fc variantat or close to the desired location of action.

The dosing amounts and frequencies of administration are, in a preferredembodiment, selected to be therapeutically or prophylacticallyeffective. As is known in the art, adjustments for protein degradation,systemic versus localized delivery, and rate of new protease synthesis,as well as the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

The concentration of the therapeutically active Fc variant in theformulation may vary from about 0.1 to 100 weight %. In a preferredembodiment, the concentration of the Fc variant is in the range of 0.003to 1.0 molar. In order to treat a patient, a therapeutically effectivedose of the Fc variant of the present invention may be administered. By“therapeutically effective dose” herein is meant a dose that producesthe effects for which it is administered. The exact dose will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques. Dosages may range from 0.0001 to 100mg/kg of body weight or greater, for example 0.1, 1, 10, or 50 mg/kg ofbody weight, with 1 to 10 mg/kg being preferred.

In some embodiments, only a single dose of the Fc variant is used. Inother embodiments, multiple doses of the Fc variant are administered.The elapsed time between administrations may be less than 1 hour, about1 hour, about 1-2 hours, about 2-3 hours, about 3-4 hours, about 6hours, about 12 hours, about 24 hours, about 48 hours, about 2-4 days,about 4-6 days, about 1 week, about 2 weeks, or more than 2 weeks.

In other embodiments the Fc variants of the present invention areadministered in metronomic dosing regimes, either by continuous infusionor frequent administration without extended rest periods. Suchmetronomic administration may involve dosing at constant intervalswithout rest periods. Typically such regimens encompass chronic low-doseor continuous infusion for an extended period of time, for example 1-2days, 1-2 weeks, 1-2 months, or up to 6 months or more. The use of lowerdoses may minimize side effects and the need for rest periods.

In certain embodiments the Fc variant of the present invention and oneor more other prophylactic or therapeutic agents are cyclicallyadministered to the patient. Cycling therapy involves administration ofa first agent at one time, a second agent at a second time, optionallyadditional agents at additional times, optionally a rest period, andthen repeating this sequence of administration one or more times. Thenumber of cycles is typically from 2-10. Cycling therapy may reduce thedevelopment of resistance to one or more agents, may minimize sideeffects, or may improve treatment efficacy.

Combination- and Co-Therapies

The Fc variants of the present invention may be administeredconcomitantly with one or more other therapeutic regimens or agents. Theadditional therapeutic regimes or agents may be used to improve theefficacy or safety of the Fc variant. Also, the additional therapeuticregimes or agents may be used to treat the same disease or a comorbidityrather than to alter the action of the Fc variant. For example, an Fcvariant of the present invention may be administered to the patientalong with chemotherapy, radiation therapy, or both chemotherapy andradiation therapy. The Fc variant of the present invention may beadministered in combination with one or more other prophylactic ortherapeutic agents, including but not limited to cytotoxic agents,chemotherapeutic agents, cytokines, growth inhibitory agents,anti-hormonal agents, kinase inhibitors, anti-angiogenic agents,cardioprotectants, immunostimulatory agents, immunosuppressive agents,agents that promote proliferation of hematological cells, angiogenesisinhibitors, protein tyrosine kinase (PTK) inhibitors, additional Fcvariants, FcγRIIb or other Fc receptor inhibitors, or other therapeuticagents.

The terms “in combination with” and “co-administration” are not limitedto the administration of said prophylactic or therapeutic agents atexactly the same time. Instead, it is meant that the Fc variant of thepresent invention and the other agent or agents are administered in asequence and within a time interval such that they may act together toprovide a benefit that is increased versus treatment with only eitherthe Fc variant of the present invention or the other agent or agents. Itis preferred that the Fc variant and the other agent or agents actadditively, and especially preferred that they act synergistically. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended. The skilled medical practitioner candetermine empirically, or by considering the pharmacokinetics and modesof action of the agents, the appropriate dose or doses of eachtherapeutic agent, as well as the appropriate timings and methods ofadministration.

In one embodiment, the Fc variants of the present invention areadministered with one or more additional molecules comprising antibodiesor Fc. The Fc variants of the present invention may be co-administeredwith one or more other antibodies that have efficacy in treating thesame disease or an additional comorbidity; for example two antibodiesmay be administered that recognize two antigens that are overexpressedin a given type of cancer, or two antigens that mediate pathogenesis ofan autoimmune or infectious disease.

Examples of anti-cancer antibodies that may be co-administered include,but are not limited to, anti 17-IA cell surface antigen antibodies suchas Panorex™ (edrecolomab); anti-4-1BB antibodies; anti-4Dc antibodies;anti-A33 antibodies such as A33 and CDP-833; anti-α4β1 integrinantibodies such as natalizumab; anti-α4β7 integrin antibodies such asLDP-02; anti-αVβ1 integrin antibodies such as F-200, M-200, and SJ-749;anti-αVβ3 integrin antibodies such as abciximab, CNTO-95, Mab-17E6, andVitaxin™; anti-complement factor 5 (C5) antibodies such as 5G1.1;anti-CA125 antibodies such as OvaRex® (oregovomab); anti-CD3 antibodiessuch as Nuvion® (visilizumab) and Rexomab; anti-CD4 antibodies such asIDEC-151, MDX-CD4, OKT4A; anti-CD6 antibodies such as Oncolysin B andOncolysin CD6; anti-CD7 antibodies such as HB2; anti-CD19 antibodiessuch as B43, MT-103, and Oncolysin B; anti-CD20 antibodies such as 2H7,2H7.v16, 2H7.v114, 2H7.v115, Bexxar® (tositumomab), Rituxan®(rituximab), Zevalin® (Ibritumomab tiuxetan), and PRO70769; anti-CD22antibodies such as Lymphocide™ (epratuzumab); anti-CD23 antibodies suchas IDEC-152; anti-CD25 antibodies such as basiliximab and Zenapax®(daclizumab); anti-CD30 antibodies such as AC10, MDX-060, and SGN-30;anti-CD33 antibodies such as Mylotarg® (gemtuzumab ozogamicin),Oncolysin M, and Smart M195; anti-CD38 antibodies; anti-CD40 antibodiessuch as SGN-40 and toralizumab; anti-CD40L antibodies such as 5c8,Antova™, and IDEC-131; anti-CD44 antibodies such as bivatuzumab;anti-CD46 antibodies; anti-CD52 antibodies such as Campath®(alemtuzumab); anti-CD55 antibodies such as SC-1; anti-CD56 antibodiessuch as huN901-DM1; anti-CD64 antibodies such as MDX-33; anti-CD66eantibodies such as XR-303; anti-CD74 antibodies such as IMMU-110;anti-CD80 antibodies such as galiximab and IDEC-114; anti-CD89antibodies such as MDX-214; anti-CD123 antibodies; anti-CD138 antibodiessuch as B-B4-DM1; anti-CD146 antibodies such as AA-98; anti-CD148antibodies; anti-CEA antibodies such as cT84.66, labetuzumab, andPentacea™; anti-CTLA-4 antibodies such as MDX-101; anti-CXCR4antibodies; antibodies such as ABX-EGF, Erbitux® (cetuximab), IMC-C225,and Merck Mab 425; anti-EpCAM antibodies such as Crucell's anti-EpCAM,ING-1, and IS-IL-2; anti-ephrin B2/EphB4 antibodies; anti-Her2antibodies such as Herceptin®, MDX-210; anti-FAP (fibroblast activationprotein) antibodies such as sibrotuzumab; anti-ferritin antibodies suchas NXT-211; anti-FGF-1 antibodies; anti-FGF-3 antibodies; anti-FGF-8antibodies; anti-FGFR antibodies, anti-fibrin antibodies; anti-G250antibodies such as WX-G250 and Rencarex®; anti-GD2 gangliosideantibodies such as EMD-273063 and TriGem; anti-GD3 gangliosideantibodies such as BEC2, KW-2871, and mitumomab; anti-gpIIb/IIIaantibodies such as ReoPro; anti-heparinase antibodies; anti-Her2/ErbB2antibodies such as Herceptin® (trastuzumab), MDX-210, and pertuzumab;anti-HLA antibodies such as Oncolym®, Smart 1D10; anti-HM1.24antibodies; anti-ICAM antibodies such as ICM3; anti-IgA receptorantibodies; anti-IGF-1 antibodies such as CP-751871 and EM-164;anti-IGF-1R antibodies such as IMC-A12; anti-IL-6 antibodies such asCNTO-328 and elsilimomab; anti-IL-15 antibodies such as HuMax™-IL15;anti-KDR antibodies; anti-laminin 5 antibodies; anti-Lewis Y antigenantibodies such as Hu3S193 and IGN-311; anti-MCAM antibodies; anti-Mudantibodies such as BravaRex and TriAb; anti-NCAM antibodies such asERIC-1 and ICRT; anti-PEM antigen antibodies such as Theragyn andTherex; anti-PSA antibodies; anti-PSCA antibodies such as IG8; anti-Ptkantbodies; anti-PTN antibodies; anti-RANKL antibodies such as AMG-162;anti-RLIP76 antibodies; anti-SK-1 antigen antibodies such as MonopharmC; anti-STEAP antibodies; anti-TAG72 antibodies such as CC49-SCA andMDX-220; anti-TGF-β antibodies such as CAT-152; anti-TNF-α antibodiessuch as CDP571, CDP870, D2E7, Humira® (adalimumab), and Remicade®(infliximab); anti-TRAIL-R1 and TRAIL-R2 antibodies; anti-VE-cadherin-2antibodies; and anti-VLA-4 antibodies such as Antegren™. Furthermore,anti-idiotype antibodies including but not limited to the GD3 epitopeantibody BEC2 and the gp72 epitope antibody 105AD7, may be used. Inaddition, bispecific antibodies including but not limited to theanti-CD3/CD20 antibody Bi20 may be used.

Examples of antibodies that may be co-administered to treat autoimmuneor inflammatory disease, transplant rejection, GVHD, and the likeinclude, but are not limited to, anti-α4β7 integrin antibodies such asLDP-02, anti-beta2 integrin antibodies such as LDP-01, anti-complement(C5) antibodies such as 5G1.1, anti-CD2 antibodies such as BTI-322,MEDI-507, anti-CD3 antibodies such as OKT3, SMART anti-CD3, anti-CD4antibodies such as IDEC-151, MDX-CD4, OKT4A, anti-CD11a antibodies,anti-CD14 antibodies such as IC14, anti-CD18 antibodies, anti-CD23antibodies such as IDEC 152, anti-CD25 antibodies such as Zenapax,anti-CD40L antibodies such as 5c8, Antova, IDEC-131, anti-CD64antibodies such as MDX-33, anti-CD80 antibodies such as IDEC-114,anti-CD147 antibodies such as ABX-CBL, anti-E-selectin antibodies suchas CDP850, anti-gpIIb/IIIa antibodies such as ReoPro/Abcixima,anti-ICAM-3 antibodies such as ICM3, anti-ICE antibodies such as VX-740,anti-FcR1 antibodies such as MDX-33, anti-IgE antibodies such asrhuMab-E25, anti-IL-4 antibodies such as SB-240683, anti-IL-5 antibodiessuch as SB-240563, SCH55700, anti-IL-8 antibodies such as ABX-IL8,anti-interferon gamma antibodies, and anti-TNFa antibodies such asCDP571, CDP870, D2E7, Infliximab, MAK-195F, anti-VLA-4 antibodies suchas Antegren. Examples of other Fc-containing molecules that may beco-administered to treat autoimmune or inflammatory disease, transplantrejection, GVHD, and the like include, but are not limited to, the p75TNF receptor/Fc fusion Enbrel® (etanercept) and Regeneron's IL-1 trap.

Examples of antibodies that may be co-administered to treat infectiousdiseases include, but are not limited to, anti-anthrax antibodies suchas ABthrax, anti-CMV antibodies such as CytoGam and sevirumab,anti-cryptosporidium antibodies such as CryptoGAM, Sporidin-G,anti-helicobacter antibodies such as Pyloran, anti-hepatitis Bantibodies such as HepeX-B, Nabi-HB, anti-HIV antibodies such asHRG-214, anti-RSV antibodies such as felvizumab, HNK-20, palivizumab,RespiGam, and anti-staphylococcus antibodies such as Aurexis, Aurograb,BSYX-A110, and SE-Mab.

Alternatively, the Fc variants of the present invention may beco-administered or with one or more other molecules that compete forbinding to one or more Fc receptors. For example, co-administeringinhibitors of the inhibitory receptor FcγRIIb may result in increasedeffector function. Similarly, co-administering inhibitors of activatingreceptors, for example FcγRIIIa, may minimize unwanted effectorfunction. Fc receptor inhibitors include but are not limited to Fcvariants that are engineered to act as competitive FcγR inhibitors, aswell as other immunoglobulins and specifically intravenousimmunoglobulin (IVIg). In one embodiment, the inhibitor is administeredand allowed to act before the Fc variant is administered. An alternativeway of achieving the effect of sequential dosing would be to provide animmediate release dosage form of the Fc receptor inhibitor and then asustained release formulation of the Fc variant of the invention. Theimmediate release and controlled release formulations could beadministered separately or be combined into one unit dosage form.Administration of an FcγRIIb inhibitor may also be used to limitunwanted immune responses, for example anti-Factor VIII antibodyresponse following Factor VIII administration to hemophiliacs.

In one embodiment, the Fc variants of the present invention areadministered with a chemotherapeutic agent. By “chemotherapeutic agent”as used herein is meant a chemical compound useful in the treatment ofcancer. Examples of chemotherapeutic agents include but are not limitedto alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan; androgenssuch as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY 117018, onapristone, and toremifene(Fareston); anti-metabolites such as methotrexate and 5-fluorouracil(5-FU); folic acid analogues such as denopterin, methotrexate,pteropterin, trimetrexate; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; folic acidreplenisher such as frolinic acid; nitrogen mustards such aschlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; platinum analogs such ascisplatin and carboplatin; vinblastine; platinum; proteins such asarginine deiminase and asparaginase; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; taxanes,e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.)and docetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France);topoisomerase inhibitor RFS 2000; thymidylate synthase inhibitor (suchas Tomudex); additional chemotherapeutics including aceglatone;aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;difluoromethylornithine (DMFO); elformithine; elliptinium acetate;etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®;razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; etoposide(VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; retinoic acid; esperamicins; capecitabine.Pharmaceutically acceptable salts, acids or derivatives of any of theabove may also be used.

A chemotherapeutic or other cytotoxic agent may be administered as aprodrug. By “prodrug” as used herein is meant a precursor or derivativeform of a pharmaceutically active substance that is less cytotoxic totumor cells compared to the parent drug and is capable of beingenzymatically activated or converted into the more active parent form.See, for example Wilman, 1986, Biochemical Society Transactions, 615thMeeting Belfast, 14:375-382; and Stella et al., “Prodrugs: A ChemicalApproach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardtet al., (ed.): 247-267, Humana Press, 1985. The prodrugs that may finduse with the present invention include but are not limited tophosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-containing prodrugs, D-aminoacid-modified prodrugs, glycosylated prodrugs, beta-lactam-containingprodrugs, optionally substituted phenoxyacetamide-containing prodrugs oroptionally substituted phenylacetamide-containing prodrugs,5-fluorocytosine and other 5-fluorouridine prodrugs which can beconverted into the more active cytotoxic free drug. Examples ofcytotoxic drugs that can be derivatized into a prodrug form for use withthe Fc variants of the present invention include but are not limited toany of the aforementioned chemotherapeutic agents.

A variety of other therapeutic agents may find use for administrationwith the Fc variants of the present invention. In one embodiment, the Fcvariant is administered with an anti-angiogenic agent. By“anti-angiogenic agent” as used herein is meant a compound that blocks,or interferes to some degree, the development of blood vessels. Theanti-angiogenic factor may, for instance, be a small molecule or aprotein, for example an antibody, Fc fusion, or cytokine, that binds toa growth factor or growth factor receptor involved in promotingangiogenesis. The preferred anti-angiogenic factor herein is an antibodythat binds to Vascular Endothelial Growth Factor (VEGF). Other agentsthat inhibit signaling through VEGF may also be used, for exampleRNA-based therapeutics that reduce levels of VEGF or VEGF-R expression,VEGF-toxin fusions, Regeneron's VEGF-trap, and antibodies that bindVEGF-R. In an alternate embodiment, the Fc variant is administered witha therapeutic agent that induces or enhances adaptive immune response,for example an antibody that targets CTLA-4. Additionalanti-angiogenesis agents include, but are not limited to, angiostatin(plasminogen fragment), antithrombin III, angiozyme, ABT-627, Bay12-9566, benefin, bevacizumab, bisphosphonates, BMS-275291,cartilage-derived inhibitor (CDI), CA1, CD59 complement fragment,CEP-7055, Col 3, combretastatin A-4, endostatin (collagen XVIIIfragment), farnesyl transferase inhibitors, fibronectin fragment,gro-beta, halofuginone, heparinases, heparin hexasaccharide fragment,HMV833, human chorionic gonadotropin (hCG), IM-862, interferon alpha,interferon beta, interferon gamma, interferon inducible protein 10(IP-10), interleukin-12, kringle 5 (plasminogen fragment), marimastat,metalloproteinase inhibitors (eg. TIMPs), 2-methodyestradiol, MMI 270(CGS 27023A), plasminogen activator inhibitor (PAI), platelet factor-4(PF4), prinomastat, prolactin 16 kDa fragment, proliferin-relatedprotein (PRP), PTK 787/ZK 222594, retinoids, solimastat, squalamine,SS3304, SU5416, SU6668, SU11248, tetrahydrocortisol-S,tetrathiomolybdate, thalidomide, thrombospondin-1 (TSP-1), TNP-470,transforming growth factor beta (TGF-β), vasculostatin, vasostatin(calreticulin fragment), ZS6126, and ZD6474.

In a preferred embodiment, the Fc variant is administered with atyrosine kinase inhibitor. By “tyrosine kinase inhibitor” as used hereinis meant a molecule that inhibits to some extent tyrosine kinaseactivity of a tyrosine kinase. Examples of such inhibitors include butare not limited to quinazolines, such as PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines,such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)-phthalimide); tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Lambert); antisensemolecules (e.g. those that bind to ErbB-encoding nucleic acid);quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No.5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering A G);pan-ErbB inhibitors such as C1-1033 (Pfizer); Affinitac (ISIS 3521;Isis/Lilly); Imatinib mesylate (STI571,Gleevec®; Novartis); PKI 166(Novartis); GW2016 (Glaxo SmithKline); C1-1033 (Pfizer); EKB-569(Wyeth); Semaxinib (Sugen); ZD6474 (AstraZeneca); PTK-787(Novartis/Schering AG); INC-1C11 (Imclone); or as described in any ofthe following patent publications: U.S. Pat. No. 5,804,396; PCT WO99/09016 (American Cyanimid); PCT WO 98/43960 (American Cyanamid); PCTWO 97/38983 (Warner-Lambert); PCT WO 99/06378 (Warner-Lambert); PCT WO99/06396 (Warner-Lambert); PCT WO 96/30347 (Pfizer, Inc); PCT WO96/33978 (AstraZeneca); PCT WO96/3397 (AstraZeneca); PCT WO 96/33980(AstraZeneca), gefitinib (IRESSA™, ZD1839, AstraZeneca), and OSI-774(Tarceva™, OSI Pharmaceuticals/Genentech).

In another embodiment, the Fc variant is administered with one or moreimmunomodulatory agents. Such agents may increase or decrease productionof one or more cytokines, up- or down-regulate self-antigenpresentation, mask MHC antigens, or promote the proliferation,differentiation, migration, or activation state of one or more types ofimmune cells. Immunomodulatory agents include but not limited to:non-steroidal anti-inflammatory drugs (NSAIDs) such as asprin,ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin,ketoralac, oxaprozin, nabumentone, sulindac, tolmetin, rofecoxib,naproxen, ketoprofen, and nabumetone; steroids (eg. glucocorticoids,dexamethasone, cortisone, hydroxycortisone, methylprednisolone,prednisone, prednisolone, triamcinolone, azulfidineicosanoids such asprostaglandins, thromboxanes, and leukotrienes; as well as topicalsteroids such as anthralin, calcipotriene, clobetasol, and tazarotene);cytokines such as TGFb, IFNa, IFNb, IFNg, IL-2, IL-4, IL-10; cytokine,chemokine, or receptor antagonists including antibodies, solublereceptors, and receptor-Fc fusions against BAFF, B7, CCR2, CCR5, CD2,CD3, CD4, CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28,CD40, CD40L, CD44, CD45, CD52, CD64, CD80, CD86, CD147, CD152,complement factors (C5, D) CTLA4, eotaxin, Fas, ICAM, ICOS, IFNα, IFNβ,IFNγ, IFNAR, IgE, IL-1, IL-2, IL-2R, IL-4, IL-5R, IL-6, IL-8, IL-9IL-12, IL-13, IL-13R1, IL-15, IL-18R, IL-23, integrins, LFA-1, LFA-3,MHC, selectins, TGFβ, TNFα, TNFβ, TNF-R1, T-cell receptor, includingEnbrel® (etanercept), Humira® (adalimumab), and Remicade® (infliximab);heterologous anti-lymphocyte globulin; other immunomodulatory moleculessuch as 2-amino-6-aryl-5 substituted pyrimidines, anti-idiotypicantibodies for MHC binding peptides and MHC fragments, azathioprine,brequinar, bromocryptine, cyclophosphamide, cyclosporine A,D-penicillamine, deoxyspergualin, FK506, glutaraldehyde, gold,hydroxychloroquine, leflunomide, malononitriloamides (eg. leflunomide),methotrexate, minocycline, mizoribine, mycophenolate mofetil, rapamycin,and sulfasasazine.

In an alternate embodiment, Fc variants of the present invention areadministered with a cytokine. By “cytokine” as used herein is meant ageneric term for proteins released by one cell population that act onanother cell as intercellular mediators. Examples of such cytokines arelymphokines, monokines, and traditional polypeptide hormones. Includedamong the cytokines are growth hormone such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-beta; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-alpha, beta, and -gamma; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosisfactor such as TNF-alpha or TNF-beta; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell culture,and biologically active equivalents of the native sequence cytokines.

In a preferred embodiment, cytokines or other agents that stimulatecells of the immune system are co-administered with an Fc variant of thepresent invention. Such a mode of treatment may enhance desired effectorfunction. For example, agents that stimulate NK cells, including but notlimited to IL-2 may be co-administered. In another embodiment, agentsthat stimulate macrophages, including but not limited to C5a, formylpeptides such as N-formyl-methionyl-leucyl-phenylalanine(Beigier-Bompadre et. al. (2003) Scand. J. Immunol. 57: 221-8), may beco-administered. Also, agents that stimulate neutrophils, including butnot limited to G-CSF, GM-CSF, and the like may be administered.Furthermore, agents that promote migration of such immunostimulatorycytokines may be used. Also additional agents including but not limitedto interferon gamma, IL-3 and IL-7 may promote one or more effectorfunctions. In an alternate embodiment, cytokines or other agents thatinhibit effector cell function are co-administered with an Fc variant ofthe present invention. Such a mode of treatment may limit unwantedeffector function.

In an additional embodiment, the Fc variant is administered with one ormore antibiotics, including but not limited to: aminoglycosideantibiotics (eg. apramycin, arbekacin, bambermycins, butirosin,dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,ribostamycin, sisomycin, spectrinomycin), aminocyclitols (eg.sprctinomycin), amphenicol antibiotics (eg. azidamfenicol,chloramphenicol, florfrnicol, and thiamphemicol), ansamycin antibiotics(eg. rifamide and rifampin), carbapenems (eg. imipenem, meropenem,panipenem); cephalosporins (eg. cefaclor, cefadroxil, cefamandole,cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide,cefpirome, cefprozil, cefuroxine, cefixime, cephalexin, cephradine),cephamycins (cefbuperazone, cefoxitin, cefminox, cefmetazole, andcefotetan); lincosamides (eg. clindamycin, lincomycin); macrolide (eg.azithromycin, brefeldin A, clarithromycin, erythromycin, roxithromycin,tobramycin), monobactams (eg. aztreonam, carumonam, and tigernonam);mupirocin; oxacephems (eg. flomoxef, latamoxef, and moxalactam);penicillins (eg. amdinocillin, amdinocillin pivoxil, amoxicillin,bacampicillin, bexzylpenicillinic acid, benzylpenicillin sodium,epicillin, fenbenicillin, floxacillin, penamecillin, penethamatehydriodide, penicillin o-benethamine, penicillin O, penicillin V,penicillin V benzoate, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium); polypeptides (eg. bacitracin, colistin,polymixin B, teicoplanin, vancomycin); quinolones (amifloxacin,cinoxacin, ciprofloxacin, enoxacin, enrofloxacin, feroxacin, flumequine,gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, moxifloxacin,nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin,pipemidic acid, rosoxacin, rufloxacin, sparfloxacin, temafloxacin,tosufloxacin, trovafloxacin); rifampin; streptogramins (eg.quinupristin, dalfopristin); sulfonamides (sulfanilamide,sulfamethoxazole); tetracyclenes (chlortetracycline, demeclocyclinehydrochloride, demethylchlortetracycline, doxycycline, duramycin,minocycline, neomycin, oxytetracycline, streptomycin, tetracycline,vancomycin).

Anti-fungal agents such as amphotericin B, ciclopirox, clotrimazole,econazole, fluconazole, flucytosine, itraconazole, ketoconazole,niconazole, nystatin, terbinafine, terconazole, and tioconazole may alsobe used.

Antiviral agents including protease inhibitors, reverse transcriptaseinhibitors, and others, including type I interferons, viral fusioninhibitors, and neuramidase inhibitors, may also be used. Examples ofantiviral agents include, but are not limited to, acyclovir, adefovir,amantadine, amprenavir, clevadine, enfuvirtide, entecavir, foscarnet,gangcyclovir, idoxuridine, indinavir, lopinavir, pleconaril, ribavirin,rimantadine, ritonavir, saquinavir, trifluridine, vidarabine, andzidovudine, may be used.

The Fc variants of the present invention may be combined with othertherapeutic regimens. For example, in one embodiment, the patient to betreated with an antibody or Fc fusion of the present invention may alsoreceive radiation therapy. Radiation therapy can be administeredaccording to protocols commonly employed in the art and known to theskilled artisan. Such therapy includes but is not limited to cesium,iridium, iodine, or cobalt radiation. The radiation therapy may be wholebody irradiation, or may be directed locally to a specific site ortissue in or on the body, such as the lung, bladder, or prostate.Typically, radiation therapy is administered in pulses over a period oftime from about 1 to 2 weeks. The radiation therapy may, however, beadministered over longer periods of time. For instance, radiationtherapy may be administered to patients having head and neck cancer forabout 6 to about 7 weeks. Optionally, the radiation therapy may beadministered as a single dose or as multiple, sequential doses. Theskilled medical practitioner can determine empirically the appropriatedose or doses of radiation therapy useful herein. In accordance withanother embodiment of the invention, the Fc variant of the presentinvention and one or more other anti-cancer therapies are employed totreat cancer cells ex vivo. It is contemplated that such ex vivotreatment may be useful in bone marrow transplantation and particularly,autologous bone marrow transplantation. For instance, treatment of cellsor tissue(s) containing cancer cells with Fc variant and one or moreother anti-cancer therapies, such as described above, can be employed todeplete or substantially deplete the cancer cells prior totransplantation in a recipient patient.

Radiation therapy may also comprise treatment with an isotopicallylabeled molecule, such as an antibody. Examples ofradioimmunotherapeutics include but Zevalin™ (Y-90 labeled anti-CD20),LymphoCide™ (Y-90 labeled anti-CD22) and Bexxar™ (I-131 labeledanti-CD20)

It is of course contemplated that the Fc variants of the invention mayemploy in combination with still other therapeutic techniques such assurgery or phototherapy.

Clinical Trial Design and Post-Approval Treatment Strategies

Pharmacogenomic approaches to clinical trials and therapy areembodiments of the present invention. A number of the receptors that mayinteract with the Fc variants of the present invention are polymorphicin the human population. For a given patient or population of patients,the efficacy of the Fc variants of the present invention may be affectedby the presence or absence of specific polymorphisms in proteins. Forexample, FcγRIIIs is polymorphic at position 158, which is commonlyeither V (high affinity) or F (low affinity). Patients with the V/Vhomozygous genotype are observed to have a better clinical response totreatment with the anti-CD20 antibody Rituxan® (rituximab) (Carton etal., 2002, Blood 99:754-758; Weng et al., 2003, J Clin Oncol21:3940-3947; Dall'Ozzo et al., 2004, Cancer Res 64:4664-9). Additionalpolymorphisms include but are not limited to FcγRIIa R131 or H131, andsuch polymorphisms are known to either increase or decrease Fc bindingand subsequent biological activity, depending on the polymorphism. Fcvariants of the present invention may bind preferentially to aparticular polymorphic form of a receptor, for example F158 FcγRIIIa, orto bind with equivalent affinity to all of the polymorphisms at aparticular position in the receptor, for example both the V158 and F158polymorphisms of FcγRIIIa. In a preferred embodiment, Fc variants of thepresent invention that provide equivalent binding to polymorphisms maybe used in an antibody to eliminate the differential efficacy seen inpatients with different polymorphisms. Such a property may give greaterconsistency in therapeutic response and reduce non-responding patientpopulations. Such variant Fc with identical binding to receptorpolymorphisms may have increased biological activity, such as ADCC, CDCor circulating half-life, or alternatively decreased activity, viamodulation of the binding to the relevant Fc receptors. In a preferredembodiment, Fc variants of the present invention may bind with higher orlower affinity to one of the polymorphisms of a receptor, eitheraccentuating the existing difference in binding or reversing thedifference. Such a property may allow creation of therapeuticsparticularly tailored for efficacy with a patient population possessingsuch polymorphism. For example, a patient population possessing anFcγRIIb polymorphism that binds with higher affinity to Fc, couldreceive a drug containing an Fc variant with reduced binding to suchpolymorphic form of the receptor, creating a more efficacious drug.

In a preferred embodiment, patients are screened for one or morepolymorphisms in order to predict the efficacy of the Fc variants of thepresent invention. This information may be used, for example, to selectpatients to include or exclude from clinical trials or, post-approval,to provide guidance to physicians and patients regarding appropriatedosages and treatment options. For example, the anti-CD20 antibodyrituximab is minimally effective in patients that are homozygous orheterozygous for F158 FcγRIIIa (Carton et al., 2002, Blood 99:754-758;Weng et al., 2003, J Clin Oncol 21:3940-3947; Dall'Ozzo et al., 2004,Cancer Res 64:4664-9). Such patients may show an improved clinicalresponse to antibodies comprising an Fc variant of the presentinvention. In one embodiment, patients are selected for inclusion inclinical trials if their genotype indicates that they are likely torespond significantly better to an antibody of the present invention ascompared to one or more currently used antibody therapeutics. In anotherembodiment, appropriate dosages and treatment regimens are determinedusing such genotype information. In another embodiment, patients areselected for inclusion in a clinical trial or for receipt of therapypost-approval based on their polymorphism genotype, where such therapycontains an Fc variant engineered to be specifically efficacious forsuch population, or alternatively where such therapy contains an Fcvariant that does not show differential activity to the different formsof the polymorphism.

Included in the present invention are diagnostic tests to identifypatients who are likely to show a favorable clinical response to an Fcvariant of the present invention, or who are likely to exhibit asignificantly better response when treated with an Fc variant of thepresent invention versus one or more currently used antibodytherapeutics. Any of a number of methods for determining FcγRpolymorphisms in humans known in the art may be used.

In a preferred embodiment, patients are screened to predict the efficacyof the Fc polypeptides of the present invention. This information may beused, for example, to select patients to include or exclude fromclinical trials or, post-approval, to provide guidance to physicians andpatients regarding appropriate dosages and treatment options. Screeningmay involve the determination of the expression level or distribution ofthe target antigen. For example, the level of Her2/neu expression iscurrently used to select which patients will most favorably respond totrastuzumab therapy. Screening may also involve determination of geneticpolymorphisms, for example polymorphisms related to FcγRs or FcαRs. Forexample, patients who are homozygous or heterozygous for the F158polymorphic form of FcγRIIIa may respond clinically more favorably tothe Fc polypeptides of the present invention. Information obtained frompatient screening may be used to select patients for inclusion inclinical trials, to determine appropriate dosages and treatmentregimens, or for other clinical applications. Included in the presentinvention are diagnostic tests to identify patients who are likely toshow a favorable clinical response to an Fc polypeptide of the presentinvention, or who are likely to exhibit a significantly better responsewhen treated with an Fc polypeptide of the present invention versus oneor more currently used biotherapeutics. Any of a number of methods fordetermining antigen expression levels, antigen distribution, and/orgenetic polymorphisms in humans known in the art may be used.

Furthermore, the present invention comprises prognostic tests performedon clinical samples such as blood and tissue samples. Such tests mayassay for effector function activity, including but not limited toopsonization, ADCC, CDC, ADCP, or for killing, regardless of mechanism,of cancerous or otherwise pathogenic cells. In a preferred embodiment,ADCC assays, such as those described herein, are used to predict, for aspecific patient, the efficacy of a given Fc polypeptide of the presentinvention. Such information may be used to identify patients forinclusion or exclusion in clinical trials, or to inform decisionsregarding appropriate dosages and treatment regimens. Such informationmay also be used to select a drug that contains a particular Fc variantthat shows superior activity in such an assay.

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation.

For all positions discussed in the present invention, numbering isaccording to the EU index or EU numbering scheme (Kabat et al., 1991,Sequences of Proteins of Immunological Interest, 5th Ed., United StatesPublic Health Service, National Institutes of Health, Bethesda), whichrefers to the numbering of the EU antibody (Edelman et al., 1969, ProcNatl Acad Sci USA 63:78-85). Those skilled in the art of antibodies willappreciate that these conventions consist of nonsequential numbering inspecific regions of an immunoglobulin sequence, enabling a normalizedreference to conserved positions in immunoglobulin families.Accordingly, the positions of any given immunoglobulin as defined by EUindex will not necessarily correspond to its sequential sequence. FIGS.3 a-3 b shows the sequential and EU index numbering schemes for theantibody alemtuzumab in order to illustrate this principal more clearly.It should also be noted that polymorphisms have been observed at anumber of Fc positions, including but not limited to Kabat 270, 272,312, 315, 356, and 358, and thus slight differences between thepresented sequence and sequences in the scientific literature may exist.

Fc variants and Fc variant libraries were designed using computational-and sequence-based methods as described in U.S. Ser. No. 10/672,280 andU.S. Ser. No. 10/822,231. Experimental libraries were designed insuccessive rounds of computational and experimental screening. Design ofsubsequent Fc libraries benefited from feedback from prior libraries,and thus typically comprised combinations of Fc variants that showedfavorable properties in the previous screen. FIG. 4 shows residues atwhich amino acid modifications were made in the Fc variants of thepresent invention, mapped onto the human Fc/FcγRIIIb structure. Theentire set of Fc variants that were constructed and experimentallytested is shown in FIGS. 41 a-41 pp.

Example 1 Molecular Biology and Protein Expression/Purification

The majority of experimentation on the Fc variants was carried out inthe context of the anti-cancer antibody alemtuzumab (Campath®, aregistered trademark of IIex Pharmaceuticals LP). Alemtuzumab binds ashort linear epitope within its target antigen CD52 (Hale et al., 1990,Tissue Antigens 35:118-127; Hale, 1995, Immunotechnology 1:175-187).Alemtuzumab has been chosen as the primary engineering template becauseits efficacy is due in part to its ability to recruit effector cells(Dyer et al., 1989, Blood 73:1431-1439; Friend et al., 1991, TransplantProc 23:2253-2254; Hale et al., 1998, Blood 92:4581-4590; Glennie etal., 2000, Immunol Today 21:403-410), and because production and use ofits antigen in binding assays are relatively straightforward. In orderto evaluate the optimized Fc variants of the present invention in thecontext of other antibodies, select Fc variants were evaluated in theanti-Her2 antibody trastuzumab (Herceptin®, a registered trademark ofGenentech), the anti-CD20 antibody rituximab (Rituxan®, a registeredtrademark of IDEC Pharmaceuticals Corporation), the anti-EGFR antibodycetuximab (Erbitux®, a registered trademark of Imclone), and theanti-CD20 antibody PRO70769 (PCT/US2003/040426, entitled “ImmunoglobulinVariants and Uses Thereof”). The use of alemtuzumab, trastuzumab,rituximab, cetuximab, and PRO70769 for screening purposes is not meantto constrain the present invention to any particular antibody.

The IgG1 full length light (V_(L)-C_(L)) and heavy (V_(H)-Cγ1-Cγ2-Cγ3)chain antibody genes for alemtuzumab (campath-1H, James et al., 1999, JMol Bio/289: 293-301), trastuzumab (hu4D5-8; Carter et al., 1992, ProcNatl Acad Sci USA 89:4285-4289; Gerstner et al., 2002, J. Mol. Biol.,321: 851-862), rituximab (C2B8, U.S. Pat. No. 6,399,061), and cetuximab(C225, PCT US96/09847) were constructed using recursive PCR withconvenient end restriction sites to facilitate subcloning. The geneswere ligated into the mammalian expression vector pcDNA3.1Zeo(Invitrogen), comprising the full length light kappa (Cκ) and heavychain IgG1 constant regions. The V_(H)-Cγ1-Cγ2-Cγ3 clone in pcDNA3.1zeowas used as a template for mutagenesis of the Fc region. Mutations wereintroduced into this clone using PCR-based mutagenesis or quick-changemutagenesis (Stratagene) techniques. Fc variants were sequenced toconfirm the fidelity of the sequence. Plasmids containing heavy chaingene (V_(H)-Cγ1-Cγ2-Cγ3) (wild-type or variants) were co-transfectedwith plasmid containing light chain gene (V_(L)-C_(L)) into 293T cells.Media were harvested 5 days after transfection. Expression ofimmunoglobulin was monitored by screening the culture supernatant oftransfectomas by western using peroxidase-conjugated goat-anti human IgG(Jackson ImmunoResearch, catalog #109-035-088). FIG. 5 shows expressionof wild-type alemtuzumab and variants 1 through 10 in 293T cells.Antibodies were purified from the supernatant using protein A affinitychromatography (Pierce, Catalog #20334. FIG. 6 shows results of theprotein purification for WT alemtuzumab. Antibody Fc variants showedsimilar expression and purification results to WT. Some Fc variants weredeglycosylated in order to determine their solution and functionalproperties in the absence of carbohydrate. To obtain deglycosylatedantibodies, purified alemtuzumab antibodies were incubated withpeptide-N-glycosidase (PNGase F) at 37° C. for 24 h. FIG. 7 presents anSDS PAGE gel confirming deglycosylation for several Fc variants and WTalemtuzumab.

In order to confirm the functional fidelity of alemtuzumab producedunder these conditions, the antigenic CD52 peptide, fused to GST, wasexpressed in E. coli BL21 (DE3) under IPTG induction. Both un-inducedand induced samples were run on a SDS PAGE gel, and transferred to PVDFmembrane. For western analysis, either alemtuzumab from Sotec (finalconcentration 2.5 ng/ul) or media of transfected 293T cells (finalalemtuzumab concentration about 0.1-0.2 ng/ul) were used as primaryantibody, and peroxidase-conjugated goat-anti human IgG was used assecondary antibody. FIG. 8 presents these results. The ability to bindtarget antigen confirms the structural and functional fidelity of theexpressed alemtuzumab. Fc variants that have the same variable region asWT alemtuzumab are anticipated to maintain a comparable binding affinityfor antigen.

The gene encoding the extracellular region of human V158 FcγRIIIa wasobtained by PCR from a clone obtained from the Mammalian Gene Collection(MGC:22630). F158 FcγRIIIa was constructed by mutagenesis of the V158FcγRIIIa gene. The genes encoding the extracellular regions of humanFcγRI, human FcγRIIa, human FcγRIIb, human FcγRIIc, mouse FcγRIII, andhuman FcRn α chain and β-microglobulin chain were constructed usingrecursive PCR. FcγRs and FcRn α chain were fused at the C-terminus witha 6×His-tag and a GST-tag. All genes were subcloned into the pcDNA3.1zeovector. For expression, vectors containing human FcγRs were transfectedinto 293T cells, FcRn α chain and β-microglobulin chain wereco-transfected into 293T cells, and mouse FcγRIII was transfected intoNIH3T3 cells. Media containing secreted receptors were harvested 3 dayslater and purified using Nickel affinity chromatography. For westernanalysis, membrane was probed with an anti-GST antibody. FIG. 9 presentsan SDS PAGE gel that shows the results of expression and purification ofhuman V158 FcγRIIIa. Purified human C1q protein complex was purchasedcommercially (Quidel Corp., San Diego).

Example 2 Fc Ligand Binding Assays

Binding to the human Fc ligands FcγRI, FcγRIIa, FcγRIIb, FcγRIIc,FcγRIIIa, C1q, and FcRn was measured for the designed Fc variants.Binding affinities were measured using an AlphaScreen™ assay (AmplifiedLuminescent Proximity Homogeneous Assay (ALPHA), PerkinElmer, Wellesley,Mass.), a bead-based luminescent proximity assay. Laser excitation of adonor bead excites oxygen, which if sufficiently close to the acceptorbead generates a cascade of chemiluminescent events, ultimately leadingto fluorescence emission at 520-620 nm. WT alemtuzumab antibody wasbiotinylated by standard methods for attachment to streptavidin donorbeads, and GST-tagged FcγRs and FcRn were bound to glutathione chelateacceptor beads. For the C1q binding assay, untagged C1q protein wasconjugated with Digoxygenin (DIG, Roche) using N-hydrosuccinimide (NHS)chemistry and bound to DIG acceptor beads. For the protein A bindingassay, protein A acceptor beads were purchased directly fromPerkinElmer. The AlphaScreen assay was applied as a competition assayfor screening Fc variants. In the absence of competing Fc variants, WTantibody and FcγR interact and produce a signal at 520-620 nm. Additionof untagged Fc variant competes with the WT FdFcγR interaction, reducingfluorescence quantitatively to enable determination of relative bindingaffinities. Fc variants were screened in the context of eitheralemtuzumab or trastuzumab, and select Fc variants were also screened inthe context of rituximab and cetuximab.

FIG. 10 shows AlphaScreen data for binding to human V158 FcγRIIIa byselect Fc variants. The binding data were normalized to the maximum andminimum luminescence signal for each particular curve, provided by thebaselines at low and high antibody concentrations respectively. The datawere fit to a one site competition model using nonlinear regression, andthese fits are represented by the curves in the figure. These fitsprovide the inhibitory concentration 50% (IC50) (i.e. the concentrationrequired for 50% inhibition) for each antibody, illustrated by thedotted lines in FIG. 10, thus enabling the relative binding affinitiesof Fc variants to be quantitatively determined. By dividing the IC50 foreach variant by that of WT alemtuzumab, the fold-enhancement orreduction relative to WT Herceptin (Fold WT) are obtained. Here, WTalemtuzumab has an IC50 of (4.63×10⁻⁹)×(2)=9.2 nM, whereas S239D has anIC50 of (3.98×10⁻¹⁰)×(2)=0.8 nM. Thus S239D alemtuzumab binds 9.2 nM/0.8nM=11.64-fold more tightly than WT alemtuzumab to human V158 FcγRIIIa.FIGS. 11 a and 11 b provide AlphaScreen data showing additional Fcvariants, with substitutions at positions 239, 264, 272, 274, and 332,that bind more tightly to FcγRIIIa, and thus are candidates forimproving the effector function of Fc polypeptides.

Fc variants were also screened in parallel for other Fc ligands. Asdiscussed, the inhibitory receptor FcγRIIb plays an important role ineffector function. Exemplary data for binding of select Fc variants ofthe invention to human FcγRIIb, as measured by the AlphaScreen, areprovided in FIG. 12. FcγRIIa is an activating receptor that is highlyhomologous to FcγRIIb. Exemplary data for binding of select Fc variantsto the R131 polymorphic form of human FcγRIIa are provided in FIG. 13.Another important Fc ligand is the neonatal Fc receptor FcRn. Asdiscussed, this receptor binds to the Fc region between the Cγ2 and Cγ3domains; because binding mediates endosomal recycling, affinity of Fcfor FcRn is a key determinant of antibody and Fc fusionpharmacokinetics. Exemplary data showing binding of select Fc variantsto FcRn, as measured by the AlphaScreen, are provided in FIG. 14. Thebinding site for FcRn on Fc, between the Cγ2 and Cγ3 domains, isoverlapping with the binding site for bacterial proteins A and G.Because protein A is frequently employed for antibody purification,select variants were tested for binding to this Fc ligand. FIG. 15provides these AlphaScreen data. Although protein A was not included inthe parallel screen for all variants, the ability of the Fc variants tobe purified using protein A chromatography (see Example 1) implies thatfor the majority of Fc variants the capacity to bind protein A, andmoreover the integrity of the Cγ2-Cγ3 hinge region, are unaffected bythe Fc substitutions.

The data for binding of Fc variants to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc,FcγRIIIa, C1q, and FcRn were analyzed as described above for FIG. 11.The fold-enhancement or reduction relative to WT for binding of eachvariant to each Fc ligand, as measured by the AlphaScreen, are providedin FIGS. 41 a-41 pp. The table presents for each variant the variantnumber (Variant), the substitution(s) of the variant, the antibodycontext (Context), the fold affinity relative to WT (Fold) and theconfidence (Conf) in the fold affinity for binding to each Fc ligand,and the IIIa:IIb specificity ratio (IIIa:IIb) (see below). Multiple datasets were acquired for many of the variants, and all data for a givenvariant are grouped together. The context of the antibody indicateswhich antibodies have been constructed with the particular Fc variant;a=alemtuzumab, t=trastuzumab, r=rituximab, c=cetuximab, and p=PRO70769.The data provided were acquired in the context of the first antibodylisted, typically alemtuzumab, although in some cases trastuzumab. Anasterix (*) indicates that the data for the given Fc ligand was acquiredin the context of trastuzumab. A fold (Fold) above 1 indicates anenhancement in binding affinity, and a fold below 1 indicates areduction in binding affinity relative to the parent antibody for thegiven Fc ligand. Confidence values (Conf) correspond to the logconfidence levels, provided from the fits of the data to a sigmoidaldose response curve. As is known in the art, a lower Conf valueindicates lower error and greater confidence in the Fold value. The lackof data for a given variant and Fc ligand indicates either that the fitsto the data did not provide a meaningful value, or that the variant wasnot tested for that Fc ligand.

FIGS. 41 a-41 pp shows that a number of Fc variants have been obtainedwith enhanced affinities and altered specificities for the various Fcligands. Some Fc variants of the present invention provide selectiveenhancement in binding affinity to different Fc ligands, whereas otherprovide selective reduction in binding affinity to different Fc ligands.By “selective enhancement” as used herein is meant an improvement in ora greater improvement in binding affinity of an Fc variant to one ormore Fc ligands relative to one or more other Fc ligands. For example,for a given variant, the Fold WT for binding to, say FcγRIIa, may begreater than the Fold WT for binding to, say FcγRIIb. By “selectivereduction” as used herein is meant a reduction in or a greater reductionin binding affinity of an Fc variant to one or more Fc ligands relativeto one or more other Fc ligands. For example, for a given variant, theFold WT for binding to, say FcγRI, may be lower than the Fold WT forbinding to, say FcγRIIb. As an example of such selectivity, G236Sprovides a selective enhancement to FcγRII's (IIa, IIb, and IIc)relative to FcγRI and FcγRIIIa, with a somewhat greater enhancement toFcγRIIa relative to FcγRIIb and FcγRIIc. G236A, however, is highlyselectively enhanced for FcγRIIa, not only with respect to FcγRI andFcγRIIIa, but also over FcγRIIb and FcγRIIc. Selective enhancements andreductions are observed for a number of Fc variants, including but notlimited to variants comprising substitutions at residues L234, L235,G236, S267, H268, R292, E293, Q295, Y300, S324, A327, L328, A330, andT335. Overall, the data provided in FIGS. 41 a-41 pp show that it isindeed possible to tune the Fc region for Fc ligand specificity, oftenby using very subtle mutational differences, despite the fact that anumber of highly homologous receptors bind to the same FcγR bindingsite. The present invention provides a number of Fc variants that may beused to selectively enhance, as well as selectively reduce, affinity ofan Fc polypeptide for certain Fc ligands relative to others. Collectionsof Fc variants such as these will not only enable the generation ofantibodies and Fc fusions that have effector function tailored for thedesired outcome, but they also provide a unique set of reagents withwhich to experimentally investigate and characterize effector functionbiology.

As discussed, optimal effector function may result from Fc variantswherein affinity for activating FcγRs is greater than affinity for theinhibitory FcγRIIb. Indeed a number of Fc variants have been obtainedthat show differentially enhanced binding to FcγRIIIa over FcγRIIb.AlphaScreen data directly comparing binding to FcγRIIIa and FcγRIIb fortwo Fc variants with this specificity profile, A330L and A330Y, areshown in FIGS. 16 a and 16 b. This concept can be defined quantitativelyas the fold-enhancement or -reduction of the activating FcγRIIIa (FIGS.41 a-41 pp, column 12) divided by the fold-enhancement or -reduction ofthe inhibitory FcγRIIb (FIGS. 41 a-41 pp, column 8), herein referred toas the “FcγRIIIa-fold:FcγRIIb-fold ratio” or “IIIa:IIb ratio”. Thisvalue is provided in column 18 of FIGS. 41 a-41 pp (as IIIa:IIb).Combination of A330L and A330Y with other variants, for exampleA330L/I332E, A330Y/I332, and S239D/A330L/I332E, provide very favorableIIIa:IIb ratios. FIGS. 41 a-41 pp shows that a number of Fc variantsprovide a positive, favorable FcγRIIIa to FcγRIIb specificity profile,with a IIIa:IIb ratio as high as 86:1.

Some of the most promising Fc variants of the present invention forenhancing effector function have both substantial increases in affinityfor FcγRIIIa and favorable FcγRIIIa-fold:FcγRIIb-fold ratios. Theseinclude, for example, S239D/I332E (FcγRIIIa-fold=56-192,FcγRIIIa-fold:FcγRIIb-fold=3), S239D/A330Y/I332E (FcγRIIIa-fold=130),S239D/A330L/I332E (FcγRIIIa-fold=139, FcγRIIIa-fold:FcγRIIb-fold=18),and S239D/S298A/I332E (FcγRIIIa-fold=295,FcγRIIIa-fold:FcγRIIb-fold=48). FIGS. 17 a-17 c show AlphaScreen datamonitoring binding of these and other Fc variants in the context oftrastuzumab to human V158 FcγRIIIa and human FcγRIIb.

In addition to alemtuzumab and trastuzumab, select Fc variants werescreened in the context of other antibodies in order to investigate thebreadth of their applicability. AlphaScreen data measuring binding ofselect Fc variants to human V158 FcγRIIIa in the context of rituximaband cetuximab are shown in FIG. 18 and FIG. 19 respectively. Togetherwith the data shown previously for alemtuzumab and trastuzumab, theresults indicate consistent binding enhancements regardless of theantibody context, and thus that the Fc variants of the present inventionare broadly applicable to antibodies and Fc fusions.

As discussed above, an important parameter of Fc-mediated effectorfunction is the affinity of Fc for both V158 and F158 polymorphic formsof FcγRIIIa. AlphaScreen data comparing binding of select variants tothe two receptor allotypes are shown in FIG. 20 a (V158 FcγRIIIa) andFIG. 20 b (F158 FcγRIIIa). As can be seen, all variants improve bindingto both FcγRIIIa allotypes. These data indicate that those Fc variantsof the present invention with enhanced effector function will be broadlyapplicable to the entire patient population, and that enhancement toclinical efficacy will potentially be greatest for the low responsivepatient population who need it most.

The FcγR binding affinities of these Fc variants were furtherinvestigated using Surface Plasmon Resonance (SPR) (Biacore, Uppsala,Sweden). SPR is a sensitive and extremely quantitative method thatallows for the measurement of binding affinities of protein-proteininteractions, and has been used to effectively measure Fc/FcγR binding(Radaev et al., 2001, J Biol Chem 276:16478-16483). SPR thus provides anexcellent complementary binding assay to the AlphaScreen assay.His-tagged V158 FcγRIIIa was immobilized to an SPR chip, and WT and Fcvariant alemtuzumab antibodies were flowed over the chip at a range ofconcentrations. Binding constants were obtained from fitting the datausing standard curve-fitting methods. Table 3 presents dissociationconstants (Kd) for binding of select Fc variants to V158 FcγRIIIa andF158 FcγRIIIa obtained using SPR, and compares these with IC50s obtainedfrom the AlphaScreen assay. By dividing the Kd and IC50 for each variantby that of WT alemtuzumab, the fold-improvements over WT (Fold WT) areobtained.

TABLE 3 SPR SPR AlphaScreen AlphaScreen V158 F158 V158 F158 FcγRIIIaFcγRIIIa FcγRIIIa FcγRIIIa Kd Fold Kd Fold IC50 Fold IC50 Fold (nM) WT(nM) WT (nM) WT (nM) WT WT 68 730 6.4 17.2 V264I 64 1.1 550 1.3 4.5 1.411.5 1.5 I332E 31 2.2 72 10.1 1.0 6.4 2.5 6.9 V264I/I332E 17 4.0 52 14.00.5 12.8 1.1 15.6 S298A 52 1.3 285 2.6 2.9 2.2 12.0 1.4 S298A/E333A/ 391.7 156 4.7 2.5 2.6 7.5 2.3 K334A

The SPR data corroborate the improvements to FcγRIIIa affinity observedby AlphaScreen assay. Table 3 further indicates the superiority ofV264I/I332E and 1332E over S298A and S298A/E333A/K334A; whereasS298A/E333A/K334A improves Fc binding to V158 and F158 FcγRIIIa by1.7-fold and 4.7-fold respectively, I332E shows binding enhancements of2.2-fold and 10.1-fold respectively, and V264I/I332E shows bindingenhancements of 4.0-fold and 14-fold respectively. Also worth noting isthat the affinity of V264I/I332E for F158 FcγRIIIa (52 nM) is betterthan that of WT for the V158 allotype (68 nM), suggesting that this Fcvariant, as well as those with even greater improvements in binding, mayenable the clinical efficacy of antibodies for the low responsivepatient population to achieve that currently possible for highresponders. The correlation between the SPR and AlphaScreen bindingmeasurements are shown in FIGS. 21 a-21 d. FIGS. 21 a and 21 b show theKd-1050 correlations for binding to V158 FcγRIIIa and F158 FcγRIIIarespectively, and FIGS. 21 c and 21 d show the fold-improvementcorrelations for binding to V158 FcγRIIIa and F158 FcγRIIIarespectively. The good fits of these data to straight lines (r²=0.9,r²=0.84, r²=0.98, and r²=0.90) support the accuracy the AlphaScreenmeasurements, and validate its use for determining the relative FcγRbinding affinities of Fc variants.

SPR data were also acquired for binding of select trastuzumab Fcvariants to human V158 FcγRIIIa, F158 FcγRIIIa, and FcγRIIb. These dataare shown in Table 4. The Fc variants tested show substantial bindingenhancements to the activating receptor FcγRIIIa, with over 100-foldtighter binding observed for interaction of S239D/I332E/S298A with F158FcγRIIIa. Furthermore, for the best FcγRIIIa binders, F158FcγRIIIa/FcγRIIb ratios of 3-4 are observed.

TABLE 4 SPR SPR SPR V158 FcγRIIIa F158 FcγRIIIa FcγRIIb Kd Fold Kd FoldIC50 Fold (nM) WT (nM) WT (nM) WT WT 363.5 503 769 V264I/I332E 76.9 4.7252 2.0 756 1.0 V264I/I332E/ 113.0 3.2 88 5.7 353 2.2 A330L S239D/I332E/8.2 44.3 8.9 56.5 46 16.7 A330L S239D/I332E/ 8.7 41.8 4.9 102.7 32 24.0S298A S239D/I332E/ 12.7 28.6 6.3 79.8 35 22.0 V264I/A330L

As discussed, although there is a need for greater effector function,for some antibody therapeutics, reduced or eliminated effector functionmay be desired. Several Fc variants in FIGS. 41 a-41 pp substantiallyreduce or ablate FcγR binding, and thus may find use in antibodies andFc fusions wherein effector function is undesirable. AlphaScreen datameasuring binding of some exemplary Fc variants to human V158 FcγRIIIaare shown in FIGS. 22 a and 22 b. These Fc variants, as well as theiruse in combination, may find use for eliminating effector function whendesired, for example in antibodies and Fc fusions whose mechanism ofaction involves blocking or antagonism but not killing of the cellsbearing target antigen. Based on the data provided in FIGS. 41 a-41 pp,preferred positions for reducing Fc ligand binding and/or effectorfunction, that is positions that may be modified to reduce binding toone or more Fc ligands and/or reduce effector function, include but arenot limited to positions 232, 234, 235, 236, 237, 239, 264, 265, 267,269, 270, 299, 325, 328, 329, and 330.

Example 3 ADCC of Fc variants

In order to determine the effect on effector function, cell-based ADCCassays were performed on select Fc variants. ADCC was measured using theDELFIA® EuTDA-based cytotoxicity assay (Perkin Elmer, Mass.) withpurified human peripheral blood monocytes (PBMCs) as effector cells.Target cells were loaded with BATDA at 1×10⁶ cells/ml, washed 4 timesand seeded into 96-well plate at 10,000 cells/well. The target cellswere then opsonized using Fc variant or WT antibodies at the indicatedfinal concentration. Human PBMCs, isolated from buffy-coat were added atthe indicated fold-excess of target cells and the plate was incubated at37° C. for 4 hrs. The co-cultured cells were centrifuged at 500×g,supernatants were transferred to a separate plate and incubated with Eusolution, and relative fluorescence units were measured using a PackardFusion™ α-FP HT reader (Packard Biosciences, IL). Samples were run intriplicate to provide error estimates (n=3, +/-S.D.). PBMCs wereallotyped for the V158 or F158 FcγRIIIa allotype using PCR.

ADCC assays were run on Fc variant and WT alemtuzumab using DoHH-2lymphoma target cells. FIG. 23 a is a bar graph showing the ADCC ofthese proteins at 10 ng/ml antibody. Results show that alemtuzumab Fcvariants I332E, V2641, and I332E/V264I have substantially enhanced ADCCcompared to WT alemtuzumab, with the relative ADCC enhancementsproportional to their binding improvements to FcγRIIIa as indicated byAlphaScreen assay and SPR. The dose dependence of ADCC on antibodyconcentration is shown in FIG. 23 b. The binding data were normalized tothe minimum and maximum fluorescence signal for each particular curve,provided by the baselines at low and high antibody concentrationsrespectively. The data were fit to a sigmoidal dose-response model usingnonlinear regression, represented by the curve in the figure. The fitsenable determination of the effective concentration 50% (EC50) (i.e. theconcentration required for 50% effectiveness), which provides therelative enhancements to ADCC for each Fc variant. The EC50s for thesebinding data are analogous to the IC50s obtained from the AlphaScreencompetition data, and derivation of these values is thus analogous tothat described in Example 2 and FIG. 11. In FIG. 23 b, the log(EC50)s,obtained from the fits to the data, for WT, V264I/I332E, and S239D/I332Ealemtuzumab are 0.99, 0.60, and 0.49 respectively, and therefore theirrespective EC50s are 9.9, 4.0, and 3.0. Thus V264I/I332E and S239E/I332Eprovide a 2.5-fold and 3.3-fold enhancement respectively in ADCC over WTalemtuzumab using PBMCs expressing heterozygous V158/F158 FcγRIIIa.These data are summarized in Table 5 below.

TABLE 5 log(EC50) EC50 (ng/ml) Fold WT WT 0.99 9.9 V264I/I332E 0.60 4.02.5 S239D/I332E 0.49 3.0 3.3

In order to determine whether these ADCC enhancements are broadlyapplicable to antibodies, select Fc variants were evaluated in thecontext of trastuzumab and rituximab. ADCC assays were run on Fc variantand WT trastuzumab using two breast carcinoma target cell lines BT474and Sk-Br-3. FIG. 24 a shows a bar graph illustrating ADCC at 1 ng/mlantibody. Results indicate that V264I and V264I/I332E trastuzumabprovide substantially enhanced ADCC compared to WT trastuzumab, with therelative ADCC enhancements proportional to their binding improvements toFcγRIIIa as indicated by AlphaScreen assay and SPR. FIGS. 24 b and 24 cshow the dose dependence of ADCC on antibody concentration for select Fcvariants. The EC50s obtained from the fits of these data and therelative fold-improvements in ADCC are provided in Table 6 below.Significant ADCC improvements are observed for 1332E trastuzumab whencombined with A330L and A330Y. Furthermore, S239D/A330L/I332E provides asubstantial ADCC enhancement, greater than 300-fold for PBMCs expressinghomozygous F158/F158 FcγRIIIa, relative to WT trastuzumab andS298A/E333A/K334A, consistent with the FcγR binding data observed by theAlphaScreen assay and SPR.

TABLE 6 log(EC50) EC50 (ng/ml) Fold WT FIG. 24b WT 1.1 11.5 I332E 0.342.2 5.2 A330Y/I332E −0.04 0.9 12.8 A330L/I332E 0.04 1.1 10.5 FIG. 24c WT−0.15 0.71 S298A/E333A/K334A −0.72 0.20 3.6 S239D/A330L/I332E −2.650.0022 323

ADCC assays were run on V264I/I332E, WT, and S298A/D333A/K334A rituximabusing WIL2-S lymphoma target cells. FIG. 25 a presents a bar graphshowing the ADCC of these proteins at 1 ng/ml antibody. Results indicatethat V264I/I332E rituximab provides substantially enhanced ADCC relativeto WT rituximab, as well as superior ADCC to S298A/D333A/K334A,consistent with the FcγRIIIa binding improvements observed byAlphaScreen assay and SPR. FIGS. 25 b and 25 c show the dose dependenceof ADCC on antibody concentration for select Fc variants. The EC50sobtained from the fits of these data and the relative fold-improvementsin ADCC are provided in Table 7 below. As can be seen S239D/I332E/A330Lrituximab provides greater than 900-fold enhancement in EC50 over WT forPBMCs expressing homozygous F158/F158 FcγRIIIa. The differences in ADCCenhancements observed for alemtuzumab, trastuzumab, and rituximab arelikely due to the use of different PBMCs, different antibodies, anddifferent target cell lines.

TABLE 7 log(EC50) EC50 (ng/ml) Fold WT FIG. 25b WT 0.23 1.7S298A/E333A/K334A −0.44 0.37 4.6 V264I/I332E −0.83 0.15 11.3 FIG. 25c WT0.77 5.9 S239D/I332E/A330L −2.20 0.0063 937

Thus far, ADCC data has been normalized such that the lower and upperbaselines of each Fc polypeptide are set to the minimal and maximalfluorescence signal for that specific Fc polypeptide, typically beingthe fluorescence signal at the lowest and highest antibodyconcentrations respectively. Although presenting the data in this matterenables a straightforward visual comparison of the relative EC50s ofdifferent antibodies (hence the reason for presenting them in this way),important information regarding the absolute level of effector functionachieved by each Fc polypeptide is lost. FIGS. 26 a and 27 b presentcell-based ADCC data for trastuzumab and rituximab respectively thathave been normalized according to the absolute minimal lysis for theassay, provided by the fluorescence signal of target cells in thepresence of PBMCs alone (no antibody), and the absolute maximal lysisfor the assay, provided by the fluorescence signal of target cells inthe presence of Triton X1000. The graphs show that the antibodies differnot only in their EC50, reflecting their relative potency, but also inthe maximal level of ADCC attainable by the antibodies at saturatingconcentrations, reflecting their relative efficacy. Thus far these twoterms, potency and efficacy, have been used loosely to refer to desiredclinical properties. In the current experimental context, however, theyare denoted as specific quantities, and therefore are here explicitlydefined. By “potency” as used in the current experimental context ismeant the EC50 of an Fc polypeptide. By “efficacy” as used in thecurrent experimental context is meant the maximal possible effectorfunction of an Fc polypeptide at saturating levels. In addition to thesubstantial enhancements to potency described thus far, FIGS. 26 a and26 b show that the Fc variants of the present invention provide greaterthan 100% enhancements in efficacy over WT trastuzumab and rituximab.

Example 4 Cross-Validation of Fc Variants

Select Fc variants were validated for their FcγR binding and ADCCimprovements in the context of two antibodies—alemtuzumab andtrastuzumab. Binding to human V158 FcγRIIIa was measured using bothAlphaScreen and SPR as described above. Exemplary AlphaScreen datameasuring FcγRIIIa binding are provided in FIG. 27. ADCC was carried outin the context of trastuzumab using Sk-Br-3 target cells and LDHdetection as described above. Exemplary ADCC data are provided in FIG.28. Table 8 provides a summary of the fold FcγRIIIa binding affinitiesto relative to WT as determined by AlphaScreen and SPR, and the foldADCC relative to WT for a series of Fc variants in the context ofalemtuzumab (alem) and trastuzumab (trast).

TABLE 8 Variant Variant Fold WT V158 FcγRIIIa Substitution NumberContext AlphaScreen SPR ADCC G236S 719 trast 2.78 1.34 0.37 G236S 719alem 6.22 6.69 S239E 43 trast 29.99 4.17 7.6 S239E 43 alem 2.64 3.28S239D 86 trast 16.9 3.5 6.1 S239D 86 alem 36.56 16.61 K246H 812 trast17.91 2.67 2 K246H 812 alem 13.58 22.36 K246Y 813 trast 17.44 2.39 1.36K246Y 813 alem 4.32 7.07 R255Y 818 trast 21.14 2.75 1.6 R255Y 818 alem0.92 1.41 E258H 820 trast 1.18 0.77 0.76 E258H 820 alem 2.35 5.5 E258Y821 trast 2.82 1.69 0.92 E258Y 821 alem 0.64 1.77 T260H 824 trast 35.322.82 T260H 824 alem 1 1.86 S267E 338 alem 9.33 2.62 H268D 350 trast45.27 4.76 4.59 H268D 350 alem 10.55 5.66 E272I 237 trast 5.86 1.63 1.38E272I 237 trast 3.24 1.99 E272R 634 alem 1.38 E272H 636 trast 1.02 0.651.28 E272H 636 alem 187.1 383.88 E272P 642 trast 0.005 0.522 0.39 E272P642 alem 1.46 1.41 E283H 839 trast 0.99 0.71 1.4 E283H 839 alem 2.31E283L 840 trast 19.88 3.68 5.2 E283L 840 alem 1.36 2.56 V284E 844 trast2.82 1.26 0.84 V284E 844 alem 1.51 E293R 555 trast 1.15 0.94 0.47 S298D364 trast 3.48 1.49 0.58 S304T 879 trast 6.33 1.65 1.02 S304T 879 alem12.85 S324I 267 trast 5.26 1.46 2.21 S324G 608 trast 3.04 1.76 3.23S324G 608 alem 13.62 14.17 K326E 103 trast 6.12 2.12 2.87 K326E 103 alem1.86 3.13 A327D 274 trast 2.44 1.31 1.04 I332E 22 trast I332D 62 trast19 2.57 5 I332D 62 alem 21.65 11.16 E333Y 284 trast 8.24 1.94 2.23 K334I285 trast 15.24 7.1 1.2 K334T 286 trast 15.73 6.79 3.14 K334F 287 trast10.46 5.82 1.92

Example 5 ADCC at Varying Target Antigen Expression Levels

A critical parameter governing the clinical efficacy of anti-cancerantibodies is the expression level of target antigen on the surface oftumor cells. Thus a major clinical advantage of Fc variants that enhanceADCC may be that it enables the targeting of tumors that express lowerlevels of antigen. In order to test this hypothesis, WT and Fc varianttrastuzumab antibodies were tested for their ability to mediate ADCCagainst different cell lines expressing varying levels of the Her2/neutarget antigen using the DELFIA EuTDA method. Four cell lines cell linesexpressing amplified to low levels of Her2/neu receptor were used,including Sk-Br-3 (1×10⁶ copies), SkOV3 (˜1×10⁵), OVCAR3(˜1×10⁴), andMCF-7 (˜3×10³ copies) (FIG. 29 a). Target cells were loaded with BATDAin batch for 25 minutes, washed multiple times with medium and seeded at10,000 cells per well in 96-well plates. Target cells were opsonized for15 minutes with various antibodies and concentrations (final conc.ranging from 100 ng/ml to 0.0316 ng/ml in ½ log steps, including notreatment control). Human PBMCs, isolated from buffy-coat and allotypedas homozygous F158/F158 FcγRIIIa were then added to opsonized cells at25-fold excess and co-cultured at 37° C. for 4 hrs. Thereafter, plateswere centrifuged, supernatants were removed and treated with Eu3+solution, and relative fluorescence units (correlating to the level ofcell lysis) were measured using a Packard Fusion™ α-FP HT reader(PerkinElmer, Boston, Mass.). The experiment was carried out intriplicates. FIG. 29 b shows the ADCC data comparing WT and Fc varianttrastuzumab against the four different Her2/neu⁺ cell lines. TheS239D/I332E and S239D/I332E/A330L variants provide substantial ADCCenhancements over WT trastuzumab at high, moderate, and low expressionlevels of target antigen. This result suggests that the Fc variants ofthe present invention may broaden the therapeutic window of anti-cancerantibodies.

Example 6 ADCC with NK Cells as Effector Cells

Natural killer (NK) cells are a subpopulation of cells present in PBMCsthat are thought to play a significant role in ADCC. Select Fc variantswere tested in a cell-based ADCC assay in which natural killer (NK)cells rather than PBMCs were used as effector cells. In this assay therelease of endogenous lactose dehydrogenase (LDH), rather than EuTDA,was used to monitor cell lysis. FIG. 30 shows that the Fc variants showsubstantial ADCC enhancement when NK cells are used as effector cells.Furthermore, together with previous assays, the results indicate thatthe Fc variants of the present invention show substantial ADCCenhancements regardless of the type of effector cell or the detectionmethod used.

Example 7 ADCP of Fc Variants

Another important FcγR-mediated effector function is ADCP. Phagocytosisof target cancer cells may not only lead to the immediate destruction oftarget cells, but because phagocytosis is a potential mechanism forantigen uptake and processing by antigen presenting cells, enhanced ADCPmay also improve the capacity of the Fc polypeptide to elicit anadaptive immune response. The ability of the Fc variants of the presentinvention to mediate ADCP was therefore investigated. Monocytes wereisolated from heterozygous V158/F158 FcγRIIIa PBMCs using a Percollgradient. After one week in culture in the presence of 0.1 ng/ml,differentiated macrophages were detached with EDTA/PBS- and labeled withthe lipophilic fluorophore, PKH26, according to the manufacturer'sprotocol (Sigma, St Louis, Mo.). Sk-Br-3 target cells were labeled withPKH67 (Sigma, St Louis, Mo.), seeded in a 96-well plate at 20,000 cellsper well, and treated with designated final concentrations of WT or Fcvariant trastuzumab. PKH26-labeled macrophages were then added to theopsonized, labeled Sk-Br-3 cells at 20,000 cells per well and the cellswere co-cultured for 18 hrs before processing cells for analysis of duallabel flow cytometry. Percent phagocytosis was determined as the numberof cells co-labeled with PKH76 and PKH26 (macrophage+Sk-Br-3) over thetotal number of Sk-Br-3 in the population(phagocytosed+non-phagocytosed) after 10,000 counts. FIG. 31 shows datacomparing WT and Fc variant trastuzumab at various antibodyconcentrations. The results indicate that the S239D/I332E/A330L variantprovides a significant enhancement in ADCP over WT trastuzumab.

Example 8 Complement Binding and Activation by Fc Variants

Complement protein C1q binds to a site on Fc that is proximal to theFcγR binding site, and therefore it was prudent to determine whether theFc variants have maintained their capacity to recruit and activatecomplement. The AlphaScreen assay was used to measure binding of selectFc variants to the complement protein C1q. The assay was carried outwith biotinylated WT alemtuzumab antibody attached to streptavidin donorbeads as described in Example 2, and using C1q coupled directly toacceptor beads. Binding data of V2641, 1332E, S239E, and V264I/I332Erituximab shown in FIG. 32 a indicate that C1q binding is uncompromised.Cell-based CDC assays were also performed on select Fc variants toinvestigate whether Fc variants maintain the capacity to activatecomplement. Alamar Blue was used to monitor lysis of Fc variant and WTrituximab-opsonized WIL2-S lymphoma cells by human serum complement(Quidel, San Diego, Calif.). The data in FIG. 32 b show that CDC isuncompromised for the Fc variants S239E, V2641, and V264I/I332Erituximab. In contrast, FIG. 32 c shows that CDC of the Fc variantS239D/I332E/A330L is completely ablated, whereas the S239D/I332E variantmediates CDC that is comparable to WT rituximab. These results indicatethat protein engineering can be used to distinguish between differenteffector functions. Such control will not only enable the generation ofFc polypeptides, including antibodies and Fc fusions, with propertiestailored for a desired clinical outcome, but also provide a unique setof reagents with which to experimentally investigate effector functionbiology.

Example 9 Enhanced B Cell Depletion in Macaques

In order to evaluate the capacity of the Fc variants to enhance effectorfunction in vivo, a pre-clinical study was carried out wherein B celldepletion was used to measure antibody cytotoxicity in cynomogus monkeys(Macaca fascicularis). Three monkeys per sample were injectedintravenously with WT or S239D/I332E rituximab antibody, with injectionsgiven once daily over days 1-4 in approximate dose ranges of 40 pg/kg(WT control) or 1, 4, 10, or 40 μg/kg (S239D/I332E and/or WT). Actualconcentrations were determined experimentally. B cell and natural killercell levels were monitored from days 5 to 28, and cell populations werecounted using flow cytometry using B cell markers CD20+ and CD40+, andnatural killer cell markers CD3−/CD16+ and CD3−/CD8+.

FIG. 33 a shows the percent of CD20+ B cells remaining in monkeys dosedwith antibodies comprising WT or S239D/I332E rituximab. The S239D/I332Evariant and WT control at the lower dosage (1.8 and 2.1 ug/kg) show thegreatest difference in B cell counts on day 5. NK cell populations weremonitored to evaluate the impact of the effector function enhancement onthis cell type; FIG. 33 b shows that the increased CD20+ B cell killingof S239D/I332E variant does not affect natural kill cell population. Thereduction in B cell level is also dose-dependant, as is shown in FIG. 33c for day 5.

Example 10 Capacity for Testing Fc Variants in Mice

Optimization of Fc to nonhuman FcγRs may be useful for experimentallytesting Fc variants in animal models. For example, when tested in mice(for example nude mice, SCID mice, xenograft mice, and/or transgenicmice), antibodies and Fc fusions that comprise Fc variants that areoptimized for one or more mouse FcγRs may provide valuable informationwith regard to clinical efficacy, mechanism of action, and the like. Inorder to evaluate whether the Fc variants of the present invention maybe useful in such experiments, affinity of select Fc variants for mouseFcγRIII was measured using the AlphaScreen assay. The AlphaScreen assaywas carried out using biotinylated WT alemtuzumab attached tostreptavidin donor beads as described in Example 2, and GST-tagged mouseFcγRIII bound to glutathione chelate acceptor beads, expressed andpurified as described in Example 2. These binding data are shown in FIG.34 a for Fc variants in the context of alemtuzumab, and in FIGS. 34 band 34 c in the context of trastuzumab. Results show that some Fcvariants that enhance binding to human FcγRIIIa also enhance binding tomouse FcγRIII. The enhancement of mouse effector function by the Fcvariants was investigated by performing the aforementioned cell-basedADCC assays using mouse rather than human PBMC's. FIG. 35 shows that theS239D/I332E/A330L trastuzumab variant provides substantial ADCCenhancement over WT in the presence of mouse immune cells. This resultindicates that the Fc variants of the present invention, or other Fcvariants that are optimized for nonhuman FcγRs, may find use inexperiments that use animal models.

Example 11 Validation of Fc Variants Expressed in CHO Cells

Whereas the Fc variants of the present invention were expressed in 293Tcells for screening purposes, large scale production of antibodies istypically carried out by expression in Chinese Hamster Ovary (CHO) celllines. In order to evaluate the properties of CHO-expressed Fc variants,select Fc variants and WT alemtuzumab were expressed in CHO cells andpurified as described in Example 1. FIG. 36 shows AlphaScreen datacomparing binding of CHO- and 293T-expressed Fc variant and WTalemtuzumab to human V158 FcγRIIIa. The results indicate that the Fcvariants of the present invention show comparable FcγR bindingenhancements whether expressed in 293T or CHO.

Example 12 Enhancement of Fc Variants in Fucose Minus Strain

Combinations of the Fc variants of the present invention with other Fcmodifications are contemplated with the goal of generating novel Fcpolypeptides with optimized properties. It may be beneficial to combinethe Fc variants of the present invention with other Fc modifications,including modifications that alter effector function or interaction withone or more Fc ligands. Such combination may provide additive,synergistic, or novel properties in Fc polypeptides. For example, anumber of methods exist for engineering different glycoforms of Fc thatalter effector function. Engineered glycoforms may be generated by avariety of methods known in the art, many of these techniques are basedon controlling the level of fucosylated and/or bisectingoligosaccharides that are covalently attached to the Fc region. Onemethod for engineering Fc glycoforms is to express the Fc polypeptide ina cell line that generates altered glycoforms, for example Lec-13 CHOcells. In order to investigate the properties of Fc variants combinedwith engineered glycoforms, WT and V209 (S239D/I332E/A330L) trastuzumabwere expressed in Lec-13 CHO cells and purified as described above. FIG.37 a shows AlphaScreen binding data comparing the binding to human V158FcγRIIIa by WT and V209 trastuzumab expressed in 293T, CHO, and Lec-13cells. The results show that there is substantial synergy between theengineered glycoforms produced by this cell line and the Fc variants ofthe present invention. The cell-based ADCC assay, shown in FIG. 37 b,supports this result. Together these data indicate that other Fcmodifications, particularly engineered glycoforms, may be combined withthe Fc variants of the present invention to generate Fc polypeptides,for example antibodies and Fc fusions, with optimized effectorfunctions.

Example 13 Aglvcosylated Fc Variants

As discussed, one goal of the current experiments was to obtainoptimized aglycosylated Fc variants. Several Fc variants providesignificant progress towards this goal. Because it is the site ofglycosylation, substitution at N297 results in an aglycosylated Fc.Whereas all other Fc variants that comprise a substitution at N297completely ablate FcγR binding, N297D/I332E has significant bindingaffinity for FcγRIIIa, shown in FIGS. 41 a-41 pp and illustrated in FIG.38. The exact reason for this result is uncertain in the absence of ahigh-resolution structure for this variant, although the computationalscreening predictions suggest that it is potentially due to acombination of new favorable Fc/FcγR interactions and favorableelectrostatic properties. Indeed other electrostatic substitutions areenvisioned for further optimization of aglycosylated Fc. FIGS. 41 a-41pp shows that other aglycosylated Fc variants such as N297D/A330Y/I332Eand S239D/N297D/I332E provide binding enhancements that bring affinityfor FcγRIIIa within as much as 0.4- and 0.8-respectively of glycosylatedWT alemtuzumab. Combinations of these variants with other Fc variantsthat enhance FcγR binding are contemplated, with the goal of obtainingaglycosylated Fc variants that bind one or more FcγRs with affinity thatis approximately the same as or even better than glycosylated parent Fc.Preferred Fc variants for enhancing Fc ligand binding and/or effectorfunction in an aglycosylated Fc polypeptide include but are not limitedto: N297D, N297D/I332E, N297D/I332D, S239D/N297D, S239D/N297D/I332E,N297D/A330Y/I332E, and S239D/N297D/A330Y/I332E. The present invention ofcourse contemplates combinations of these aglycosylated variants withother Fc variants described herein which also enhance Fc ligand bindingand/or effector function.

An additional set of promising Fc variants provide stability andsolubility enhancements in the absence of carbohydrate. Fc variants thatcomprise substitutions at positions 241, 243, 262, and 264, positionsthat do not mediate FcγR binding but do determine the interface betweenthe carbohydrate and Fc, ablate FcγR binding, presumably because theyperturb the conformation of the carbohydrate. In deglycosylated form,however, Fc variants F241E/F243R/V262E/V264R, F241E/F243Q/V262T/V264E,F241R/F243Q/V262T/V264R, and F241E/F243Y/V262T/V264R show strongerbinding to FcγRIIIa than in glycosylated form, as shown by theAlphaScreen data in FIG. 39. This result indicates that these are keypositions for optimization of the structure, stability, solubility, andfunction of aglycosylated Fc. Together these results suggests thatprotein engineering can be used to restore the favorable functional andsolution properties of antibodies and Fc fusions in the absence ofcarbohydrate, and pave the way for aglycosylated antibodies and Fcfusions with favorable solution properties and full functionality thatcomprise substitutions at these and other Fc positions.

Example 14 Preferred Variants

Taken together, the data provided in the present invention indicate thatFc variants that provide optimized FcγR binding properties also provideenhanced effector function. Substitutions at a number of positions,including but not limited to 236, 239, 246, 246, 249, 255, 258, 260,264, 267, 268, 272, 274, 281, 283, 304, 324, 326, 327, 330, 332, 333,334, and 334 provide promising candidates for improving the effectorfunction and therefore the clinical properties of Fc polypeptides,including antibodies and Fc fusions. Because combinations of Fc variantsof the present invention have typically resulted in additive orsynergistic binding improvements, and accordingly additive orsynergistic enhancements in effector function, it is anticipated that asyet unexplored combinations of the Fc variants provided in FIGS. 41 a-41pp will also provide favorable results. Preferred Fc variants of thepresent invention for enhancing Fc ligand binding and/or effectorfunction are provided in Table 9.

TABLE 9 G236S S239D/I332E S239D/K246H/I332E S239D/K246H/T260H/I332EG236A S239D/G236A S239D/V264I/I332E S239D/K246H/H268D/I332E S239DS239D/G236S S239D/S267E/I332E S239D/K246H/H268E/I332E S239E S239D/V264IS239D/H268D/I332E S239D/H268D/S324G/I332E S239N S239D/H268DS239D/H268E/I332E S239D/H268E/S324G/I332E S239Q S239D/H268ES239D/S298A/I332E S239D/H268D/K326T/I332E S239T S239D/S298AS239D/S324G/I332E S239D/H268E/K326T/I332E K246H S239D/K326ES239D/S324I/I332E S239D/H268D/A330L/I332E K246Y S239D/A330LS239D/K326T/I332E S239D/H268E/A330L/I332E D249Y S239D/A330YS239D/K326E/I332E S239D/H268D/A330Y/I332E R255Y S239D/A330IS239D/K326D/I332E S239D/H268E/A330Y/I332E E258Y I332E/V264IS239D/A327D/I332E S239D/S298A/S267E/I332E T260H I332E/H268DS239D/A330L/I332E S239D/S298A/H268D/I332E V264I I332E/H268ES239D/A330Y/I332E S239D/S298A/H268E/I332E S267E I332E/S298AS239D/A330I/I332E S239D/S298A/S324G/I332E H268D I332E/K326ES239D/K334T/I332E S239D/S298A/S324I/I332E H268E I332E/A330LS239D/S298A/K326T/I332E E272Y I332E/A330Y S239D/S298A/K326E/I332E E272II332E/A330I S239D/S298A/A327D/I332E E272H I332E/G236AS239D/S298A/A330L/I332E K274E I332E/G236S S239D/S298A/A330Y/I332E G281DI332D/V264I S239D/K326T/A330Y/I332E E283L I332D/H268DS239D/K326E/A330Y/I332E E283H I332D/H268E S239D/K326E/A330Y/I332E S304TI332D/S298A S239D/K326E/A330L/I332E S324G I332D/K326E S324I I332D/A330LK326T I332D/A330Y A327D I332D/A330I A330Y I332D/G236A A330L I332D/G236SA330I I332D I332E I332N I332Q E333Y K334T K334F

This list of preferred Fc variants is not meant to constrain the presentinvention. Indeed all combinations of the any of the Fc variantsprovided in FIGS. 41 a-41 pp are embodiments of the present invention.Furthermore, combinations of any of the Fc variants of the presentinvention with other discovered or undiscovered Fc variants may alsoprovide favorable properties, and these combinations are alsocontemplated as embodiments of the present invention. Finally, it isanticipated from these results that other substitutions at positionsmutated in present invention may also provide favorable bindingenhancements and specificities, and thus substitutions at all positionsin FIGS. 41 a-41 pp are contemplated.

Example 15 Therapeutic Application of Fc Variants

A number of Fc variants described in the present invention havesignificant potential for improving the therapeutic efficacy ofanticancer antibodies. For illustration purposes, a number of Fcvariants of the present invention have been incorporated into thesequence of the antibody rituximab. The WT rituximab light chain andheavy chain, described in U.S. Pat. No. 5,736,137, are provided in FIGS.40 a and 40 b. The improved anti-CD20 antibody sequences are provided inFIG. 40 c. The improved anti-CD20 antibody sequences comprise at leastnon-WT amino acid selected from the group consisting of X₁, X₂, X₃, X₄,X₅, X₆, X₇, X₈, and X₉. These improved anti-CD20 antibody sequences mayalso comprise a substitution Z₁ and/or Z₂. The use of rituximab here issolely an example, and is not meant to constrain application of the Fcvariants to this antibody or any other particular Fc polypeptide.

Table 10 depicts the positions of human Fc, the wild type residue, andthe variants (SEQ ID NO: 8) that are included in particular embodimentsof the invention. Table 10 is based on IgG1, although as will beappreciated by those in the art, the same thing can be done to any Ig,particularly IgG2, IgG3 and IgG4.

TABLE 10 Position Wild Type (Human) Variants including wild type 118-220FX see FIG. 3a Vb(221) D D, K, Y Vb(222) K K, E, Y Vb(223) T T, E, KVb(224) H H, E, Y Vb(225) T T, E, K, W Fx(226) WT C Vb(227) P P, E, G,K, Y Vb(228) P P, E, G, K, Y Fx(229) (OPEN)(WT) C Vb(230) P P, A, E, G,Y Vb(231) A A, E, G, K, P, Y Vb(232) P P, E, G, K, Y Vb(233) E A, D, F,G, H, I, K, L, M, N, Q, R, S, T, V, W, Y Vb(234) L L, A, D, E, F, G, H,I, K, M, N, P, Q, R, S, T, V, W, Y Vb(235) L L, A, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, Y Vb(236) G G, A, D, E, F, H, I, K, L, M, N,P, Q, R, S, T, V, W, Y Vb(237) G G, D, E, F, H, I, K, L, M, N, P, Q, R,S, T, V, W, Y Vb(238) P P, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V,W, Y Vb(239) S S, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, YVb(240) V V, A, I, M, T Vb(241) F F, D, E, L, R, S, W, Y Fx(242) WT LVb(243) F F, E, H, L, Q, R, W, Y Vb(244) P P, H Vb(245) P P, A Vb(246) KK, D, E, H, Y Vb(247) P P, G, V Vb(248) WT K Vb(249) D D, H, Q, YFx(250-254) WT -(T-L-M-I-S)- Vb(255) R R, E, Y Fx(256-257) WT -(T-P)-Vb(258) E E, H, S, Y Fx(259) WT V Vb(260) T T, D, E, H, Y Fx(261) WT CVb(262) V V, A, E, F, I, T Vb(263) V V, A, I, M, T Vb(264) V V, A, D, E,F, G, H, I, K, L, M, N, P, Q, R, S, T, W, Y Vb(265) D D, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, W, Y Vb(266) V V, A, I, M, T Vb(267) S S, D,E, F, H, I, K, L, M, N, P, Q, R, T, V, W, Y Vb(268) H H, D, E, F, G, I,K, L, M, N, P, Q, R, T, V, W, Y Vb(269) E E, F, G, H, I, K, L, M, N, P,R, S, T, V, W, Y Vb(270) D D, F, G, H, I, L, M, P, Q, R, S, T, W, YVb(271) A A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y Vb(272) EE, D, F, G, H, I, K, L, M, P, R, S, T, V, W, Y Vb(273) V V, I Vb(274) KK, D, E, F, G, H, L, M, N, P, R, T, V, W, Y Vb(275) F F, L, W Vb(276) NN, D, E, F, G, H, I, L, M, P, R, S, T, V, W, Y Fx(277) WT W Vb(278) Y Y,D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W Fx(279) WT V Vb(280) D D,G, K, L, P, W Vb(281) G G, D, E, K, N, P, Q, Y Vb(282) V V, E, G, K, P,Y Vb(283) E E, G, H, K, L, P, R, Y Vb(284) V V, D, E, L, N, Q, T, YVb(285) H H, D, E, K, Q, W, Y Vb(286) N N, E, G, P, Y Fx(287) WT AVb(288) K K, D, E, Y Fx(289) WT T Vb(290) K K, D, H, L, N, W Vb(291) PP, D, E, G, H, I, Q, T Vb(292) R R, D, E, T, Y Vb(293) E E, F, G, H, I,L, M, N, P, R, S, T, V, W, Y Vb(294) E E, F, G, H, I, K, L, M, P, R, S,T, V, W, Y Vb(295) Q Q, D, E, F, G, H, I, M, N, P, R, S, T, V, W, YVb(296) Y Y, A, D, E, G, H, I, K, L, M, N, Q, R, S, T, V Vb(297) N N, D,E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y Vb(298) S S, D, E, F, H,I, K, M, N, Q, R, T, W, Y Vb(299) T T, A, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, V, W, Y Vb(300) Y Y, A, D, E, G, H, K, M, N, P, Q, R, S, T,V, W Vb(301) R R, D, E, H, Y Vb(302) V V, I Vb(303) V V, D, E, Y Vb(304)S S, D, H, L, N, T Vb(305) V V, E, T, Y Fx(306-312) WT-(L-T-V-L-H-Q-D)- * Vb(313) W W, F Fx(314-316) WT -(L-N-G)- Vb(317) K K,E, Q Vb(318) E E, H, L, Q, R, Y Fx(319) WT Y Vb(320) K K, D, F, G, H, I,L, N, P, S, T, V, W, Y Fx(321) WT C Vb(322) K K, D, F, G, H, I, P, S, T,V, W, Y Vb(323) V V, I Vb(324) S S, D, F, G, H, I, L, M, P, R, T, V, W,Y Vb(325) N N, A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, YVb(326) K K, I, L, P, T Vb(327) A A, D, E, F, H, I, K, L, M, N, P, R, S,T, V, W, Y Vb(328) L L, A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V,W, Y Vb(329) P P, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, YVb(330) A A, E, F, G, H, I, L, M, N, P, R, S, T, V, W, Y Vb(331) P P, D,F, H, I, L, M, Q, R, T, V, W, Y Vb(332) I I, A, D, E, F, H, K, L, M, N,P, Q, R, S, T, V, W, Y Vb(333) E E, F, H, I, L, M, N, P, T, Y Vb(334) KK, F, I, L, P, T Vb(335) T T, D, F, G, H, I, L, M, N, P, R, S, V, W, YVb(336) I I, E, K, Y Vb(337) S S, E, H, N

All references are herein expressly incorporated by reference.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

1-2. (canceled)
 3. A method of treatment comprising contacting effectorcells expressing FcγRIIb with a polypeptide, wherein said polypeptidebinds with greater affinity to the FcγRIIb receptor than a parentpolypeptide and increases the inhibitory activity of the FcγRIIbreceptor, and wherein said polypeptide comprises an Fc variant of saidparent Fc polypeptide and wherein said Fc variant comprises an aminoacid substitution S267E as compared to said parent Fc polypeptide,wherein said numbering is according to the EU index.
 4. The methodaccording to claim 3, wherein said Fc variant has an affinity forFcγRIIb receptor that is more than 5-fold greater than that of saidparent Fc polypeptide.
 5. The method according to claim 3, wherein saidFc variant has an affinity for FcγRIIb receptor that is between 5-foldand 300-fold greater than that of said parent Fc polypeptide.
 6. Themethod according to claim 3, wherein said polypeptide is an antibody. 7.The method according to claim 6, wherein said antibody is selected fromthe group consisting of a human antibody, a humanized antibody, and achimeric antibody.
 8. The method according to claim 7, wherein saidantibody is a humanized antibody.
 9. The method according to claim 7,wherein said antibody is a chimeric antibody.
 10. The method accordingto claim 6, wherein said antibody further comprises an engineeredglycoform.
 11. The method according to claim 3, wherein increasinginhibitory activity of the FcγRIIb receptor is used to treat autoimmunedisease.
 12. The method according to claim 3, wherein increasinginhibitory activity of the FcγRIIb receptor is used to treatinflammatory disease.
 13. A method of treatment comprising contactingeffector cells expressing FcγRIIb with a polypeptide, wherein saidpolypeptide binds with greater affinity to the FcγRIIb receptor than aparent polypeptide and increases the inhibitory activity of the FcγRIIbreceptor, and wherein said polypeptide comprises an Fc variant of saidparent Fc polypeptide and wherein said Fc variant comprises an aminoacid substitution S267D as compared to said parent Fc polypeptide,wherein said numbering is according to the EU index.
 14. The methodaccording to claim 13, wherein said Fc variant has an affinity forFcγRIIb receptor that is more than 5-fold greater than that of saidparent Fc polypeptide.
 15. The method according to claim 13, whereinsaid Fc variant has an affinity for FcγRIIb receptor that is between5-fold and 300-fold greater than that of said parent Fc polypeptide. 16.The method according to claim 13, wherein said polypeptide is anantibody.
 17. The method according to claim 16, wherein said antibody isselected from the group consisting of a human antibody, a humanizedantibody, and a chimeric antibody.
 18. The method according to claim 17,wherein said antibody is a humanized antibody.
 19. The method accordingto claim 17, wherein said antibody is a chimeric antibody.
 20. Themethod according to any one of claim 16, wherein said antibody furthercomprises an engineered glycoform.
 21. The method according to claim 13,wherein increasing inhibitory activity of the FcγRIIb receptor is usedto treat autoimmune disease.
 22. The method according to claim 13,wherein increasing inhibitory activity of the FcγRIIb receptor is usedto treat inflammatory disease.
 23. A method of enhancing the binding ofan Fc polypeptide to the FcγRIIb receptor, said method comprisingsubstituting in a parent Fc polypeptide the amino acid Ser at position267 with the amino acid Glu to provide an Fc variant of the parent Fcpolypeptide, wherein said numbering is according to the EU index. 24.The method according to claim 23, wherein the polypeptide is anantibody.
 25. The method according to claim 24, wherein said antibody isselected from the group consisting of a human antibody, a humanizedantibody, and a chimeric antibody.
 26. The method according to claim 24,wherein said antibody further comprises an engineered glycoform.
 27. Themethod according to claim 24, wherein the amino acid substitution ismade by generating a nucleic acid that encodes said variant.
 28. Amethod of enhancing the binding of an Fc polypeptide to the FcγRIIbreceptor, said method comprising substituting in a parent Fc polypeptidethe amino acid Ser at position 267 with the amino acid Asp to provide anFc variant of the parent Fc polypeptide, wherein said numbering isaccording to the EU index.
 29. The method according to claim 28, whereinthe polypeptide is an antibody.
 30. The method according to claim 29,wherein said antibody is selected from the group consisting of a humanantibody, a humanized antibody, and a chimeric antibody.
 31. The methodaccording to claim 29, wherein said antibody further comprises anengineered glycoform.
 32. The method according to claim 29, wherein theamino acid substitution is made by generating a nucleic acid thatencodes said variant.